Cellular Responses against DNA Damaged by Platinum Anticancer Drugs by Yongwon Jung

Cellular Responses against DNA Damaged by Platinum Anticancer Drugs by Yongwon Jung M.Sc., Chemistry Korea Advanced Institute of Technology and Science, 2000 SUBMITTED TO THE DEPARTMENT OF CHEMISTRY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BIOLOGICAL CHEMISTRY AT THE MASSACHUSETTS INSTITUTE OF TECHNOLOGY September 2005 © Massachusetts Institute of Technology, 2005 All rights reserved Signature of Author: I U fI J I Department of Chemistry August 22, 2005 a Certified by rm _ -1-x =- __ I __ '% I - ' ' Stephen J. Lippard Arthur Amos Noyes Professor of Chemistry Thesis Supervisor ) Accepted by: Robert W. Field Chairman, Departmental Committee on Graduate Studies IMASSACHUSETTS INSTITUrTE OF TECHNOLOGY OCT 1 2005 1 LIBRARIES ARCHIVES 2 This doctoral thesis has been examined by a Committee of the Department of Chemistry as follows: JoAnne Stubbe Novartis Professor of Chemistry Committee Chairman ' - (Q'ephenJ.Lippard Arthur Amos Noyes Professor of Chemistry Thesis Supervisor Catherine L. Drennan Associat herine L. Drennan Associate Professor of Chemistry al- 3 Cellular Responses against DNA Damaged by Platinum Anticancer Drugs By Yongwon Jung Submitted to the Department of Chemistry on August 16, 2005 In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Chemistry ABSTRACT The anticancer activity of platinum-based drugs such as cisplatin, carboplatin, and oxaliplatin is mediated by their ability to attack DNA such that generated adducts trigger numerous cellular responses. A better understanding of these processes is critical for developing more effective therapeutic approaches, which can increase the anti-cancer activity of the drugs while minimizing side effects and extending successful treatment to a wider range of human cancers. Chapter 1 provides the current comprehension of early cellular responses to platinum-DNA adducts. The event primarily occurs through platinum-DNA adduct recognition by a number of cellular proteins. Among proteins that recognize platinum-DNA lesions, one class constitutes proteins that selectively recognize severely distorted DNA generated by the platinum adduct formation. The TATA-binding protein (TBP) and high mobility group protein HMGB1, both highly abundant and vital proteins, are reported to bind cisplatindamaged DNA and to be involved in mediating the cytotoxic activity of platinum-based agents. Chapters 2 and 3 discuss the structural and kinetic properties of both proteins binding to platinated DNA. The TBP protein recognizes the TATA box element of transcriptional promoters and recruits other initiation factors. TBP binds with high affinity (Kd= 0.3 nM) to DNA containing site-specific cisplatin 1,2-intrastrand d(GpG) 4 cross-links. The ko and koffvalues for the formation of these TBP complexes are 1-3 x 105 M-ls-1 and -1-5 x 104 sec -l, respectively, similar to the corresponding values for the formation of a TBP-TATAbox complex. When TBP was added to an in vitro nucleotide excision repair (NER) assay, it specifically shielded cisplatin-modified 1,2-(GpG) intrastrand cross-links from repair. HMGB1, a highly conserved non-histone DNA- binding protein, interacts with specific DNA structural motifs such as those encountered at cisplatin damage, four-way junctions, and supercoils. The full-length HMGB1 protein binds to DNA containing a 1,2-intrastrand d(GpG) cross-link mainly through domain A with a dissociation constant Kd of 120 nM. Interaction of the C- terminal tail with the rest of the HMGB1 protein was examined by EDC cross-linking experiments. The acidic tail mainly interacts with domain B and linker regions rather than domain A in HMGB1. Another group of proteins that encounter platinum-damaged DNA are involved with DNA function and therefore are in frequent contact with DNA. DNA, RNA polymerases and histone proteins inevitably run into platinum-DNA adducts. Transcription inhibition by DNA adducts of cisplatin is considered to be one of the major routes by which this anticancer drug kills cancer cells. Stalled RNA polymerases at platinum-DNA lesions evoke various cellular responses such as nucleotide excision repair, polymerase degradation, and apoptosis. The consequences of RNA polymerase blockage by platinum lesions are discussed in the next two chapters. T7 RNA polymerase and site-specifically platinated DNA templates immobilized on a solid support were used. Polymerase action is inhibited at multiple sites in the vicinity of the platinum lesion. The stalled polymerase can be dissociated from the DNA by subsequent polymerases initiated from the same template. The immediate consequences of human RNA polymerase (Pol) II arrest at the site of DNA damaged by cisplatin were 5 studied in whole cells and cell extracts, with a particular focus on the stability of stalled Pol II and its subsequent ubiquitylation. Pol II was completely blocked by a cisplatin intrastrand cross-link and the stalled polymerase was quite stable in nuclear extracts as well as in cisplatin-treated HeLa cells. The stalled Pol II proteins were transcriptionally active and capable of resuming transcription beyond the DNA adduct following its chemical removal from the template. A series of experiments revealed that lysines other than Lys-48 of ubiquitin are involved in Pol II ubiquitylation following DNA damage. Only a fraction of ubiquitylated Pol II dissociates from damage sites and that it is rapidly destroyed by proteosomes. In the final chapter, hapten-conjugated platinum compounds were studied in an effort to follow DNA damaged by platinum agents in living cells. Platinum complexes containing a desthiobiotin moiety (DTB-Pt)with different linkers were synthesized and characterized in vitro and in cells. DNA damaged by DTB-Pt was strongly interacted with streptavidin-coated beads. Moreover, less than 1 fmol of DTB-Pt DNA adduct can be detected by using a simple dot blot analysis. Thesis Supervisor: Stephen J. Lippard Title: Arthur Amos Noyes Professor of Chemistry 6 This thesis is dedicated to my family. 7 Acknowledgments For the past five years I have dreamed about writing acknowledgments of my thesis and I still cannot believe I am doing it right now! There are so many people I want to thank for their contribution to this thesis. First, I need to thank my advisor, Steve Lippard. I feel very fortunate to have the opportunity to work in his lab filled with wonderful people. Steve has always trusted me and provided me the infinite freedom and an extra laboratory funding to explore bioinorganic chemistry. He is an amazing scientist who puts a great amount of effort to educate his students, which I will always respect. I would also like to thank Professor JoAnne Stubbe for helpful discussions as my committee chair, and Professor Cathy Drennan for her guidance to be a crystallographer. I joined the Lippard lab with Katie Barnes, Emily Carson, and Sungho Yoon, forming the best class ever in this lab history! Emily has been always a leader of this group. I think she does not know how much I admire her for everything she did. Katie has claimed as my "older American sister" since I have four Korean sisters already. I really appreciate that she has taken care of me in countless ways from the beginning. And there is my big brother Sungho. He is the best basketball player I have ever played with and is also the best inorganic chemist as well! We have been called as "The twin" in the basketball court. It has been a pleasure to get to know every former and current member of the Cisplatin subgroup. Seth Cohen and Yuji Mikata helped me to start working in the lab. Min Wei always encouraged me when I was (seldom) depressed. I wish my very best for Alissa Dangel, Olga Burenkova, Ariel Haskel and Sumi Mukhopadhyay. Christiana Zhang was the happiest person in our subgroup and Dong Wang was the best photographer. I am confident that Evan Guggenheim, Big Evan, will take good care of our subgroup with help from Katie Lovejoy, Datong Song, and Dong Xu after Katie and I leave. Dong, you can do it! I have also had the opportunity to mentor two great MIT undergraduates Sarah Simmons and Cindy Yuan. I wish them good luck in following their dream. I was fortunate to work with many incredible people over years in the Lippard lab. From Dongwhan Lee, Scott heldebrand, Josh Farrell, Matt Clark, Adna Ambundo, Lisa Chatwood to Liz Nolan, Andy Tennyson, Jeremy Kodanko, Rayane Moreira, and 8 Simone Friedle, it is amazing how a single lab can have these many awesome people. Especially my great baymates deserve a huge amount of thanks. Erik Dill, Viviana Izzo, and Mike McCormick are now new bosses in MY biobay. I hope Jessica Blazyk will find new home some time soon in other than Boston. Liz Cadieux is the nicest person I have ever met and she will be the best Mom as well. And finally to Matt Sazinsky, thanks for having meals and going on many trips with me, and just being my friend. I was also lucky to have two Korean lab mates, Mihee Lim and Yoojin Kim. I will not forget many nights we had Korean food together. I cannot talk about my life at MIT without all the sports I played. First, I want to thank to my IM basketball crew: Chris Chang, Leslie Murray, and Laurance Beauvais. We did make playoffs! Almost all lab members played ice hockey with Jane Kuzelka as our captain. We needed to play D- league. D league is too competitive. Playing softball with Brian Wong, Todd Harrop, and many others was great. And after five years of playing, I think I became a real member of the Lippard lab volleyball team at last. There are also many non-Lippard lab friends to whom I am grateful for assistance and support, without which I would never be able to survive this far. One of my Korean classmates, Seungjib Choi, has been an invaluable teacher and friend for me. We must have had literally thousands cups of coffee together. I also need to thank to many of my basketball friends for five years although I still don't know some of their names. Finally, but not the least, Yoon-aa Choi has made my last year at MIT so much fun and filled it with happiness. She always supported me through the tough times by giving me confidence, even when she also tried to figure out a demanding graduate life at MIT. Most of all she helped me to index this thesis for 2 hours! Well I already said this thesis is dedicated to my family. But I will try a little more here to express my appreciation to them. My Mom and Dad have always believed in me doing whatever I wanted with endless support. I know how much you care about me and pray for me. And to my four sisters, thanks for your consistent trust and just about everything you did for your so called "only brother". I am blessed with this great family. I promise I will a better person so you can be proud of me. I will be back soon! 9 Table of Contents Abstract ....................................................................................................... 3 Dedication.............................. ........................................................................ Acknowledgments............................................................................................ 6 Table of Contents ......................................................... List of Tables .......................................................... 15 List of Schemes ............................................................ 16 List of Figures ....................................................... .. 17 Glossary of Terms ......................................................... 19 Chapter 1. Direct Cellular Responses to Platinum-Induced DNA Damage ........... ............... 21 Background and Focus............................................................... 22 Biological Target of Platinum-based Anticancer Drugs ......................................... 23 Cellular uptake and efflux of platinum complex ......................................... 24 DNA: primary target for platinum drug .................................................... 25 The nature of platinum-DNA adducts ....................................................... Platinum Adducts Interaction with DNA Function Proteins ................................... 26 27 The effects of platinum adduct interaction with DNA polymerase ................... 27 The effects of platinum adduct interaction with RNA polymerase .................. 30 The effects of platinum adduct on chromatin: nucleosome structure and modification .................................................................... Repair of Platinum-damaged DNA ........................................................ 32 34 Nucleotide excision repair ................................................................. 34 Mismatch repair ................................................................. 37 DNA recombination .................................................................. 39 Proteins Binding To Platinum-damaged DNA .................................................... Repair proteins ............................................................... 39 40 NER proteins .................................................................. 40 Mismatch repair proteins ........................................................... 42 DNA-PK .................................................................. 42 HMG domain proteins .................................................................. 44 HMGB1.................................................................... 45 10 SSR P 1 .................................................................. ..................... Other Cellular Proteins .......................................................................... TBP................... ................... 47 49 ........... . ......................... 49 p53.................................................................... 49 YB-1 .................................................................. 52 PA RP-1 . . ...... .. ..................................................... 51 ....................... Concluding Remarks ................................................................. References ....................................... 52 .................................... 54 Chapter 2. Kinetic Studies of the TATA-Binding Protein Interaction with Cisplatin-Modified DN A ........................................................................................................... 80 Introduction 81 . . . . . ...... ....................................................................................... Experimental Procedures .................................................................. Preparation of Yeast TBP......... ......... ......... 83 .......................................83 Preparation of Oligonucleotides Probes ................. ...................83 Electrophoretic Mobility Shift Assay (EMSA)............................................. 84 Kinetic EMSA Analysis ............................................... 84 ................... Competition Assay.................................................................. 85 Excision Repair Assay................... 8..................6...... Footprinting Assay .................................................................. 86 Results.................................................................. ...................................... 87 Kinetics of TBPBinding to the TATA Box and Cisplatin-Damaged DNA ......... 87 Flanking Sequence Preference of TBP Binding to Cisplatin-Damaged DNA ......88 Cisplatin-Damaged DNA Sequesters TBP from the TATA Box ...................... 89 TBP Blocks NER of the Cisplatin 1,2-d(GpG) Cross-Link ............................... 89 TBPBinding Mode to Cisplatin-Damaged DNA .......................................... 90 Discussion ................... ... ...... ........ ...............................................................90 TBPBinding to Cisplatin-Damaged DNA .................................................. 90 Flanking Sequence Dependence of TBP Binding to Cisplatin-Damaged DNA...92 Biological Implications of TBP Binding to Cisplatin-Damaged DNA ............... 94 Acknowledgements ................................................................. 96 R eferences 97 . ... . . .. .. ......................................................................................... 11 Chapter 3. The Nature of Full-Length HMGB1 Binding to Cisplatin-Modified DNA ............. 116 Introduction ........................................................... 117 Experimental Procedures ........................................................... 119 Construction of Expression Vectors ........................................................ 119 Expression and Purification of HMGB1 Proteins ........................................ 119 Preparation of Oligonucleotides Probes ................................................... 120 Electrophoretic Mobility Shift Assay (EMSA)............................................ 120 Footprinting Assay ................................................................. 121 EDC Cross-Linking ................................................................. 121 Results .......... ................................................. 122 Footprinting Analysis of HMG Box Protein Binding to Cisplatin-Modified DNA......... .. ................................................. 122 Mutation of Intercalating Residues in HMGB1........................................... 122 Effect of the C-terminal Acidic Tail ......................................................... 123 Discussion .................................................................. 125 Full-Length HMGB1 Binding to Cisplatin-Modified DNA; Two Tandem HMG Boxes.................................................................. 125 The C-terminal Tail in HMGB1............................................................. 127 Implications for the Mechanism of HMGB1 Function ................................. 129 Roles of HMGB1 in Cisplatin Action ....................................................... 131 Conclusion ................................................................... References ................................................................. 132 133 Chapter 4. Multiple States of Stalled T7 RNA Polymerase at DNA Lesions Generated by Platinum Anticancer Agents ............................................................... 144 Introduction ........................................................... 145 Experimental Procedures ................................................................... 146 Materials .................................................................. 146 Construction of Site-Specifically Platinated DNA Templates ........................ Promoter-Dependent In Vitro Transcription ............................................. 147 147 12 Promoter-Independent In Vitro Transcription ........................................... 148 Multi-Round In Vitro Transcription ......................................................... 149 Platinum Removal by Cyanide Ion Treatment ........................................... 149 Results ................................................................. 150 Promoter-Dependent In Vitro Transcription on Platinated Templates ............ 150 Transcription Bypass Through Platinum Binding Sites............................... 151 Promoter-Independent In Vitro Transcription ........................................... 151 UTP-Specific Incorporation by T7 RNA Polymerase at the Site of a Cisplatin 1,2- Intrastrand d(GpG) Cross-Link ............................ ......... .........153 Multi-Round In Vitro Transcription ..................................................... 154 Restarting Transcription Following Platinum Removal by Cyanide Ion Treatment ............................................................ Discussion ................................................................. 154 155 Promoter-Dependent and -Independent Transcription Inhibition at Platinum Cross-Links .................................................................. 155 A Closer Look at Polymerase Blockage by Platinum Adducts ....................... 157 The Effect of Multiple Polymerases at the Site of a Platinum-DNA Cross- Link..................................................... 160 Resumption of Transcription of T7 RNA Polymerase Stalled at a Platinum Binding Site ............................................................ 161 Conclusion ............................................................ 162 Acknowledgments ................................................................. References .................................................................. 163 165 Chapter 5. RNA Polymerase II Blockage by Platinum DNA Damage: Polyubiquitylation of Stalled Polym erase ..................................................................................... 179 Introduction ........................................................... 180 Experimental Procedures ........................................................... 182 Materials ................................................................... 182 Construction of DNA Templates .......................................................... Preparation of HeLa Nuclear Extract ....................................................... In Vitro Transcription Assays in a HeLa Nuclear Extract ............................. 183 184 185 13 In Vitro Ubiquitylation Assays in a HeLa Nuclear Extract ........................... 186 Cellular Protein Fractionation . . ............................................................. HA-tagged Ubiquitin Expression in HeLa Cells........................................ 187 188 Immunoprecipitation. 189 Results .. .......................................................................... .... ............... ...... ..... .... ............................ 190 Stability of RNA Polymerase II Stalled at a Platinum-DNA Lesion ............... 190 Dynamic State of Stalled RNA Polymerase II: Backtracking and Transcription Resumption . ........ .... ... . ............................................... 193 Polyubiquitylation of Stalled RNA Polymerase II: In Vitro Ubiquitylation ......194 Polyubiquitylation of Stalled RNA Polymerase II in HeLa Cells. Discussion ........................ ... .. ...................196 ............................................. 99 Stability of RNA Polymerase II Stalled at a Platinum DNA Lesion................. 199 Dynamic State of Stalled RNA Polymerase II: Backtracking and Transcription Resumption ............................................................... Polyubiquitylation of Stalled RNA Polymerase II . 200 ... ................................... 201 Polyubiquitylation of Stalled RNA Polymerase II: Effect on Pol II .................. 203 Conclusion ............................ ................................... 204 Acknowledgement....................................................................................... 205 References...................................... 206 .. ... ............................................ Chapter 6. Following Cisplatin: Hapten-Conjugated Platinum Complex.............................. 220 Introduction.......................... ..................................... 221 Experimental Procedures ............................................................................. 222 Desthiobiotinamido-hexylamine-Boc (DTB6Nboc) (1) (Scheme 6.1) . Desthiobiotinamido-hexylamine (DTB6N) (2). Diboc-aminoethyl-Gly ............... 222 .......................................... 222 (3)................... 2.............................. 223 DTB9diamine (4)................................................................................ 223 DTB9Pt (5)........................................................................................ 224 Cbz-6-amino-hexanol (6) (Scheme 6.2) .................................................... 224 Cbz-6-amino-hexanal (7) ...................................................................... 225 Cbz-6-amino-hexyl-diamine-diboc (8). . ................................................... 225 DTB6diamine (9)............................................................... 225 14 DTB6Pt (10) ................................................................... 1, 4-Diazidobutane (11) (Scheme 6.3) ....................................................... 1, 4-Azidoaminobutane Pt4N 3 (12) ................................................................. (13) ....................................................... DNA Blot and Desthiobiotin Detection .................................................... Cytotoxicity Assay ................................................................. Results and Discussion .................................................................. 226 226 227 227 227 228 228 Synthesis of Desthiobiotin-Conjugated Platinum Agents ............................. 228 ....... 229 Characterization of Desthiobiotin-Conjugated Platinum Agents ......... Synthesis of Azide-Conjugated Platinum Agent ......................................... 231 Conclusion .................................................................. ...................................................... ...... Acknowledgements References ................................................................. 232 232 233 Biographical Note ............................................................. Curriculum Vitae ................................................................... 239 240 15 List of Tables Table 1.1. Key human proteins that bind to cisplatin-modified DNA ......................... Table 2.1. Duplex DNA probes and abbreviations .............................................. 72 102 Table 2.2. Calculated and observed (ESI-MS) molecular weights for platinated oligonucleotides .............................................................. 103 Table 2.3. Kinetic and thermodynamic parameters for TBPbinding to each probe .....104 Table 3.1. Affinities of HMGB1 Proteins Toward Cisplatin-Modified DNA ............... 135 Table 4.1. The complete sequences of DNA and RNA fragments employed in this study.............................................................. Table 6.1. IC50 values (M) for cisplatin, DTB9Pt, and DTB6Pt ......... 169 ................... 234 16 List of Schemes Scheme 6.1. Synthesis of DTB9Pt ............................................................... Scheme 6.2. Synthesis of DTB6Pt......... .............. ....... Scheme 6.3. Synthesis of Pt4N 3................................................................ 235 ...... 236 237 17 List of Figures Figure 1.1. Cisplatin and related platinum-based anticancer drugs ........................... 73 Figure 1.2. Platinum DNA adducts: formation and structures .................................. 74 Figure 1.3. Schematic representation of transcription inhibition by platinum lesions and consequent outcomes .................................................................. 75 Figure 1.4. The effect of platinum damage on chromatin structure and function ........ 76 Figure 1.5. The mechanism of nucleotide excision repair of platinum lesions .............. 77 Figure 1.6. The roles of proteins binding to platinated DNA in cisplatin anticancer 78 action............................................................................ Figure 1.7. Direct cellular responses to platinum adducts: overall picture of current understanding .................................................................... 79 Figure 2.1. EMSA experiment to determine kon.................................................... 105 Figure 2.2. EMSA experiment to determine koff. 106 ....................................... Figure 2.3. EMSA of TATAMLP and cisplatin-damaged probes with TBP ............. 107 Figure 2.4. Kinetic EMSA data for cisplatin-damaged probes binding to TBP.............108 Figure 2.5. Competition EMSA assay between TGGC and TATAMLP...................... 109 Figure 2.6. Competition EMSA assay between AGGA and TATAMLP..................... 110 Figure 2.7. Effect of TBP on excision repair assay of cisplatin-DNA intrastrand crosslinks in HeLa cell extract ............................................................... 111 Figure 2.8. Hydroxyl radical footprint of TBP..................................................... 113 Figure 2.9. Schematic diagram of TBP-DNA complex interactions as revealed by 115 .............................................................. footprinting Figure 3.1. Schematic representation of HMGB1 and sequence of duplex DNA probes and abbreviations ............................................................. 136 Figure 3.2. Footprint analysis of the interaction between HMGB1 proteins and 35AGGA ................................................................... 137 Figure 3.3. EMSA analysis of HMGB1 and AB165 binding to 25TGGA .................... 138 Figure 3.4. EMSA analysis of HMGB1 and AB165mutants binding to 25TGGA.........139 Figure 3.5. Footprint analysis of the interaction between HMGB1 mutant proteins and 35AGGA .................................................................. Figure 3.6. CNBr cleavage analysis of EDC cross-linked HMGB1......... Figure 3.7. EDC cross-linking of HMGB1 bound to cisplatin-modified 140 ..............141 DNA............142 Figure 3.8. EMSA analysis of cross-linked HMGB1 samples binding to 25TGGA....... 143 18 170 Figure 4.1. Construction of DNA templates ........................................................ Figure 4.2. Gel-electrophoresis analysis of promoter-dependent in vitro transcription .................................................................... 171 Figure 4.3. Gel-electrophoresis analysis of transcription bypass ............................. 172 Figure 4.4. Gel-electrophoresis analysis of promoter-independent transcription ......... 173 Figure 4.5. Analysis of T7 RNA polymerase blockage by various platinum adducts .................................................................... 174 Figure 4.6. Analysis of nucleotide incorporation by T7 RNA polymerase .................. 175 Figure 4.7. Gel-electrophoresis analysis of transcription by multiple polymerases .....176 ....... 177 Figure 4.8. Gel-electrophoresis analysis of platinum removal from DNA ...... Figure 4.9. Structures of the template strand of a T7 RNA polymerase elongation complex .................................................................. Figure 5.1. Construction of the DNA template ............................ 178 ...................209 Figure 5.2. The complete sequences of DNA fragments employed to construct 99-95 DNA.................................................................. 210 Figure 5.3. Schematic representation of expression vector construction for HA-tagged ubiquitin .................................................................. 211 Figure 5.4. Gel electrophoresis analysis of in vitro transcription in HeLa nuclear extracts .................................................................. 212 213 Figure 5.5. Western blot analysis of RNA polymerase II ........................................ Figure 5.6. Gel electrophoresis analysis of in vitro transcription in HeLa nuclear extract .................................................................. 214 Figure 5.7. Western blot analysis of in vitro ubiquitylation of RNA polymerase II......215 Figure 5.8. Western blot analysis of ubiquitylation of RNA polymerase II in HeLa cells................................................................ 216 Figure 5.9. The effect of MG132 on ubiquitylation of RNA polymerase II in HeLa cells.................................................................. 217 Figure 5.10. Subcellular localization of ubiquitylated RNA polymerase II in HeLa cells................................... ........................... 218 Figure 5.11. Model depicting the consequences of RNA polymerase II blockage by 219 cisplatin adducts ................................................................ Figure 6.1. DNA blot analysis .............................................................. 238 19 Abbreviations 1,2-d(GpG) 1,2-d(ApG) AAG AAS AdML bp BSA carboplatin cisplatin CNBr CPD CS CTR DACH DCC DNA-PK DNP DSB DTB IDTT IEDC EDTA EMSA en ERCC1 ESI-MS FACT FBS GGR HA HM(G IC 5 0 MMR MTT NER oxaliplatin PARP PBS PCNA cis-[Pt(NH 3 ) 2 {d(GpG)-N7(1 )-N7(2)}] cis-[Pt(NH3) 2 {d (ApG)-N7(1)-N7(2) }] 3-methyladenine DNA glycosylase atomic absorption spectroscopy adenovirus major late base pair bovine serum albumin cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II) cis-diamminedichloroplatinum(II) cyanogen bromide cyclobutane pyrimidine dimmer cockayne syndrome copper transporter diaminocyclohexane dicyclohexylcarbodiimide DNA-dependent protein kinase dinitrophenyl double strand break desthiobiotin dithiothreitol 1-ethyl-3(3-dimethylaminopropyl)carbodiimide ethylenediaminetetraacetic acid electrophoretic mobility shift assay ethylene diamine excision repair cross complementation group 1 electrospray ionization mass spectrometry facilitates chromatin transcription fetal bovine serum global genome repair hemagglutinin high mobility group concentration required to kill 50% of treated cells mismatch repair [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] nucleotide excision repair (1R,2R-diaminocyclohexane)oxalatoplatinum(II) poly(ADP-ribose)polymerase phosphate buffered saline proliferating cell nuclear antigen Pol II RNA polymerase II PMSF phenylmethylsulfonylfluoride polyvinylidene fluoride Replication protein A structure-specific recognition proteins 1 TATA binding protein transcription coupled repair translesion synthesis PVDF FRPA SSRP1 TBP TCR TLS 20 transplatin tsHMG UV-DRB XP YB-1 trans-diamminedichloroplatinum(II) testis specific HMG UV-damage recognition protein xeroderma pigmentosum Y-box binding protein 21 Chapter 1 Direct Cellular Responses To Platinum-Induced DNA Damage 22 BACKGROUND AND FOCUS Since the serendipitous discovery of the anti-cancer activity of cisplatin, the drug has been used extensively in cancer chemotherapy (1). Platinum-based drugs such as cisplatin, carboplatin, and oxaliplatin, are widely used against various solid tumors including ovarian, cervical, bladder, and nonsmall cell lung cancer (2). Cisplatin is particularly effective in the treatment of testicular cancer with a cure rate of over 90% and nearly 100% when tumors are discovered early (3). The clinical use of cisplatin, however, is restricted by dose-limiting side effects including nephrotoxicity, emetogenesis and neurotoxicity (4). Moreover, many tumor cells are inherently resistant or often acquire the resistance to platinum-based drugs, which further limits the use of these drugs (5). For over three decades, continuous efforts have been made to alleviate these limitations with a primary focus on the development of new platinum drugs. Over 3000 platinum compounds have been synthesized and tested for their biological activity. Of these, however, less than 30 compounds have entered clinical trials (6). Development of new anticancer platinum drugs has encountered difficulties to overcome the drawbacks of cisplatin in actual clinical tests. At present, only four platinum drugs are registered as marketed drugs and only one compound (oxaliplatin) has been approved by the FDA since cisplatin (Figure 1.1)(5,7,8). A better understanding of cellular responses to platinum drugs is being sought not only to develop novel platinum-based anticancer agents but also to find more effective cancer therapies with existing drugs. It is now generally accepted that DNA is the main biological target of cisplatin. The complicated mechanism of the anticancer action of cisplatin includes cellular uptake and transport of the drug to the nucleus, DNA adduct formation, and adduct interaction with damage-response proteins (9). Subsequent signal transduction pathways activated by this interaction between 23 platinum-DNA damage and cellular proteins lead to cell-cycle arrest and repair of DNA damage, the result of the process deciding the fate of treated cells (10). Knowledge of the precise mechanism of cisplatin action is still lacking. In particular, there is a considerable gap in our understanding of how platinum-DNA damage initiates various cellular signaling pathways. The present review focuses on the current comprehension of early cellular responses to platinum-DNA adducts. The event primarily occurs through platinumDNA adduct recognition by a number of cellular proteins, which triggers many signaling pathways (11). Proteins that encounter platinum-DNA lesions can be divided into two classes. One class comprises proteins that selectively recognize severely distorted DNA generated by the adduct formation with platinum agents. The other group of proteins are involved with DNA function and therefore are in frequent contact with DNA. Hence, these proteins, such as DNA and RNA polymerases and histone proteins, inevitably encounter platinum-DNA adducts. Here we provide recent information available for the interactions of platinum-DNA adducts with cellular proteins and the effects of the adducts on proteins that are involved in various DNArelated processes. The topics discussed will offer a useful guidance to link platinum- DNA damage with subsequent cellular pathways, and will ultimately provide a valuable basis for the development of better therapeutic strategies with platinum-based agents (12,13). Other aspects of cellular processes mediating cisplatin cytotoxicity or developing resistance against the drug are reviewed elsewhere (14-16). BIOLOGICAL TARGET OF PLATINUM-BASED ANTICANCER DRUGS DNA has been a main biological target of platinum-based anticancer drugs. These drugs generate many kinds of DNA adducts. Moreover, various platinum agents 24 display different adduct profiles, due to their unique structural and kinetic properties for DNA binding. Understanding the nature of these platinum adducts is important to successfully discuss how these adducts are recognized and processed by cellular proteins. Cellular uptake and efflux of platinum complex. Upon administration to the bloodstream, cisplatin maintains a relatively stable neutral state, due to a high concentration of chloride ion (100 mM), until the drug enters the cell. Passive diffusion has long been considered as a main mechanism for cisplatin uptake. The argument is supported by the fact that the platinum concentration is the rate limiting factor for drug accumulation inside cells, and the uptake is not saturable (17-19). In addition, the uptake of cisplatin is not inhibited by its structural analogues (20). Alternatively, however, some evidence indicates a role of active transporters for cisplatin uptake and efflux (20).For example, multiple studies demonstrated that reactive aldehydes inhibit cisplatin accumulation in cells, possibly by modifying membrane proteins (21,22).Very recently, a series of studies have indicated the direct linkage between copper transporters and the uptake and efflux of platinum compounds (23). The first evidence was the association of copper-transporting P-type adenosine triphosphatase (ATP7B),a key player in copper homeostasis, with cisplatin resistance (24).The direct connection of copper transporter to the uptake of cisplatin, however, was first discovered in a transposon mutagenesis experiment in yeast (25). Yeast cells lacking copper uptake protein Ctrl show increased resistance to cisplatin and decreased accumulation of the drug. The same results are also observed in mouse embryo fibroblast cells. Furthermore, a later study confirmed that Ctrl mediates the uptake of other platinum drugs including cisplatin analogues (26). More studies with ATP7B as well as ATP7A, 25 another copper transporting protein, suggested that the proteins modulate cisplatin levels in cells, presumably by provoking drug efflux (27-30). It is evident now that proteins managing copper homeostasis participate in regulating sensitivity of platinumbased drugs, likely through controlling the platinum level in the cell (31-34). More studies are needed to fully understand how the cellular level of platinum drug is managed by passive diffusion and copper homeostasis proteins, or possibly by other unidentified transporters. DNA: primary target for platinum drug. Once cisplatin enters the cell, the low concentration of chloride ion (4 mM) facilitates hydrolysis of the compound to produce [Pt(NH3 )Cl(OH2)]+, which is an active form of the drug (14). This cationic mono-aquated platinum compound reacts with various cellular components including proteins, RNA, DNA, membrane phospholipids, microfilaments, and thiol-containing molecules. Controlling cisplatin hydrolysis and transporting activated cisplatin to biological targets are among the key elements to be appreciated to comprehend the mechanism of action of the drug and these subjects have been extensively discussed elsewhere (35). It is now generally accepted that DNA is the primary target of cisplatin among many poteintial cellular targets. Cisplatin-treated bacteria show phenotypes that are characteristic of those treated by DNA-damaging agents (9). More conclusive proof came from the experiments with DNA repair-deficient cells (36,37). Cells lacking in DNA repair are more sensitive to cisplatin. In addition, levels of platinum atoms bound to proteins and RNA are too low to exhibit significant inhibitory effects on the targets (38). There is some evidence, however, that non-DNA targets are involved in drug action to some extent (5). 26 The nature of platinum-DNA adducts. The reaction of cisplatin with cellular components is proposed to be controlled kinetically rather than thermodynamically. This hypothesis explains the fact that cisplatin binds to DNA in the nucleus instead of reacting with S-donor ligands such as glutathione and methionine, which form more stable platinum complexes (35). Mono-aquated cisplatin [Pt(NH3 )CI(OH2)] + (tl/2 of formation reaction: -2 h) readily modifies DNA through binding to the N7 atom of a guanine or adenine base to form a monofunctional adduct (tl/2 -0.1 h) (9,39).The second chloride ligand is hydrolyzed with a half life of -2h, and eventually a bifunctional adduct (intra- or interstrand cross-link) is formed. Adducts analysis of purified DNA treated with cisplatin or DNA isolated from cisplatin-treated patients demonstrates that damaged DNA contains approximately 65% 1,2-d(GpG), 25% 1,2-d(GpA), and 5-10% 1,3-d(GpNpG) intrastrand cross-links as major forms (11). A small percentage of interstrand cross-links and monofunctional adducts is also found. Transplatin, a clinically inactive isomer of cisplatin (Figure 1.1), is unable to form a 1,2 intrastrand cross-link, owing to its stereochemistry. Platinum drugs such as carboplatin and oxaliplatin contain different leaving ligands from the chloride ions of cisplatin and therefore exhibit different kinetics for DNA binding and generate disparate adduct profiles from cisplatin (8). The formation of cisplatin adducts significantly alters the structure of target DNA. Early biochemical studies demonstrated unwinding and bending of DNA as well as duplex destabilization induced by cisplatin lesions (40,41). Detailed structures of platinum adducts have been extensively studied (9,42). Structures of duplex DNA containing 1,2- and 1,3-intrastrand cross-links are illustrated in Figure 1.2 (43-45).Major platinum adducts (intrastrand cross-links) unwind the duplex DNA in the vicinity of the platinum site, bending it towards the major groove, and generate a widened and 27 shallow minor groove. On the other hand, the interstrand DNA cross-link formed by cisplatin affords a helix bending towards the minor groove with the platinum moiety located in the minor groove. Although these platinum adducts display some degree of structural similarity, it is clear that each adduct distorts the duplex DNA in a distinct way. In addition, the structures of DNA adducts formed by platinum drugs with carrier ligands different from the amines of cisplatin show some variations from the discussed structures of cisplatin-DNA adducts (8). Different platinum adducts are believed to be distinctly recognized and processed by cellular proteins, suggesting also different roles in mediating cisplatin cytotoxicity. PLATINUM ADDUCTS INTERACTION WITH DNA FUNCTION PROTEINS Once the platinum drug forms a DNA adduct, it interferes with essential DNA functions. The inhibition of replication and transcription is a key cytotoxic ability of the drug. Events that occur following the interaction of a platinum adduct with DNA polymerases and RNA polymerases, however, contribute to more than just cytotoxicity of cisplatin. In addition, recent studies discovered the influence of cisplatin adducts on the properties of chromatin. The effects of platinum adduct interaction with DNA polymerase. The inhibition of DNA synthesis by cisplatin was discovered early and believed to contribute to the cytotoxicity of cisplatin. The activities of partially purified human DNA polymerases a and 3 are inhibited by cisplatin treatment of the DNA template (46). Cisplatin-induced inhibition of DNA replication is also observed in vivo in green monkey CV-1 cells transfected with SV40 chromosome (47). More detailed studies on the abilities of 28 different platinum adducts to block various DNA polymerases followed (48,49).Most bifunctional adducts, intra and interstrand cross-links, effectively inhibit DNA polymerases, while monofunctional adducts cannot block the polymerases. T4 and T7 DNA polymerases, DNA polymerase I and III are blocked by platinum adducts, bypassing the lesion only -10% of the time (48). Despite the evident inhibition of DNA synthesis by cisplatin based on these reports, murine leukemia L1210 cells treated with cisplatin progress through the S phase of the cell cycle and are blocked only in the G2 phase (50).DNA replication continues even in the cells that do not divide. Furthermore, a study with Chinese hamster ovary cell lines both proficient and deficient for DNA excision repair demonstrated that the inhibition of DNA synthesis is dependent only on the concentration of cisplatin and not on the sensitivity of the cell line to the drug (51). Only the content of cells arrested in the G2 phase correlates with cell line sensitivity to cisplatin. It is likely that direct inhibition of DNA replication by cisplatin-DNA damage cannot fully explain the unique properties of this anticancer agent (52). Mammalian cells possess the ability to synthesize DNA while ignoring various DNA lesions. The process, called translesion synthesis (TLS),demands specialized DNA polymerases, which are less stringent than the major replicative DNA polymerases and can accommodate damaged bases (53). In eukaryotes, the Y-family DNA polymerases (, , K, and Rev 1) and DNA polymerase , a member of the B family, perform DNA replication across DNA lesions (54). Translesion synthesis through cisplatin-DNA adducts has been an interesting aspect of DNA synthesis in cisplatin-treated cells due to its closer correlation to drug sensitivity (55). Cisplatin-resistant cells exhibit more of TLS than drug-sensitive cells (56-59). The process also has a critical role in the mutagenic property of cisplatin because of the nature of TLS, which carries out both error-prone and error-free DNA synthesis (54).The mutagenicity of cisplatin is closely related to the 29 evolution of resistance of cell lines against the drug. In fact, the reduced ability to replicate cisplatin-damaged DNA decreases the rate at which the cells obtain resistance to cisplatin. For example, suppression of human DNA polymerase involved in TLS, such as polymerase Rev 1 (60) or C (61), increases the sensitivity of cells to cisplatin and reduces the rate of appearance of cisplatin resistance at the population level. DNA polymerases that are shown to bypass replication across cisplatin adducts in vitro, include DNA polymerase 13, t, and 1, while polymerases a, , and are unable to perform TLS past platinum adducts (62-65).Each DNA polymerase displays a distinct specificity in its lesion-bypass property, such as the bypass ability, fidelity, and extension ability. For example, DNA polymerase rl bypasses platinum adducts most efficiently in error-free TLS, which was proved both in vivo and in vitro (59,66). Polymerase t is the most error-prone enzyme, mediating mainly frame-shift mutations (67). Currently, the identities of DNA polymerases that are responsible for TLS past platinum adducts in vivo are not clear. Moreover, two DNA polymerases often work together to complete TLS (54). Although polymerase ~ is unable to bypass certain DNA lesions including those from platinum agents, the enzyme has the ability to extend TLS once nucleotides are inserted opposite DNA adducts by other polymerases (54,61). Immediate cellular responses to a stalled replication fork at the site of platinumDNA lesion are still unclear. Recent studies indicate that proliferating cell nuclear antigen (PCNA) plays a key role for the TLS process by recruiting TLS DNA polymerases to the site of stalled replication forks (53). It was proposed that, following replication fork blockage, Radl8 binds to exposed single-stranded DNA at the fork and together with Rad6 mediates mono-ubiquitylation of PCNA. Mono-ubiquitylated PCNA then physically interacts with a TLS DNA polymerase to recruit and replace it 30 with the stalled replicative DNA polymerase. The efficiency and fidelity of TLS depends on the nature of the adduct and the recruited polymerase. Oxaliplatin has different properties towards replication bypass in its platinated DNA from cisplatin both in vivo and in vitro, presumably due to its bulky diaminocyclohexane (DACH) carrier ligand (Figure 1.1) (42). This behavior of oxaliplatin is thought to contribute to its distinct anticancer activity compared to cisplatin. The effects of platinum adduct interaction with RNA polymerase. Early in vitro studies reported the ability of cisplatin adducts to inhibit transcription elongation by various RNA polymerases including wheat germ RNA polymerase II (Pol II) and E. coli, T7, and SP6 RNA polymerases (68,69).Similar to the inhibition of DNA synthesis, RNA polymerases are strongly blocked by bifunctional adducts and not by mono-functional adducts. Direct transcription inhibition by cisplatin and transplatin is observed in human and hamster cell lines that are transfected with a plasmid containing a reporter gene and pre-modified by platinum compounds (70,71). A higher level of transplatin adducts is required to inhibit transcription to the same degree as cisplatin adducts. Accumulated data indicate a close relation between transcription inhibition by cisplatin and the drug's anticancer activity. RNA polymerases are believed to encounter platinum lesions at a relatively early stage in the DNA damage-response process. Approximately 100 copies of RNA polymerase I are constantly transcribing the rRNA gene in the cell (72). Although the inhibition of RNA polymerase I by platinum adducts has not been directly studied, it is speculated that the damage can block this polymerase (73). RNA polymerase II transcribes most eukaryotic genes and is one of the most abundant proteins with -300,000 copies in a single cell (74). A photobleaching experiment revealed that 25% of 31 this enzyme is persistently associated with cellular DNA to generate mRNA (75,76). RNA polymerase II has been a major focus of the field studying cellular responses to DNA damage including those by platinum drugs because of its dual roles in the process. Arrested polymerase at the site of platinum lesion not only functions as a damage recognition factor, triggering transcription-coupled repair (TCR) (77), but also mediates programmed cell death (78). Our knowledge of cisplatin adduct-induced blockage of RNA polymerase II has been greatly advanced over the past several years. The DNA probes containing a sitespecific platinum lesion are employed in in vitro transcription assays with human cell ,extracts or partially purified human transcription factors (79,80). Platinum 1,2-(GpG) and 1,3-(GpTpG)intrastrand cross-links strongly block the elongation complex. A study 'with T7 RNA polymerase revealed that polymerase action is inhibited at multiple sites in the vicinity of the platinum lesion, the nature of which can be altered by the concentration of NTPs and types of platinum adducts (81). The elongation complex is able to proceed into the site of platinum damage, where the polymerase inserts an incorrect nucleotide UTP, rather than a correct nucleotide CTP, opposite a cisplatin 1,2(GpG) cross-link. The fate of stalled RNA polymerase II at a platinum lesion is also closely examined, which can provide a useful insight into the mechanism of TCR. Solidphase in vitro transcription experiments have been employed in multiple studies (80,82). Stalled polymerases are fairly stable but can be released from DNA in an ATPdcependent manner by cellular release factors including human release factor 2 (HuF2) (73,80,83).A differently designed in vitro experiment indicated that a considerable level of stalled polymerase II proteins can remain strongly associated with damaged DNA in cell extracts (82). This result was supported by a cell fractionation experiment using cisplatin-treated HeLa cells, which demonstrated an increased level of chromatin- 32 associated polymerase II proteins following DNA damage. These polymerases are able to backtrack from the damage sites, cleave the transcripts, and re-elongate. Various cellular proteins, including CSB and TFIIS, are thought to mediate this process (Figure 1.3) (80,84).Nucleotide excision repair (NER)can occur in vitro at the DNA damage site with polymerase remaining on the DNA (83). In mammalian cells, cisplatin treatment facilitates RNA polymerase II degradation following ubiquitylation of the protein (82,85,86). In vitro transcription experiments with a cisplatin-damaged plasmid also demonstrate ubiquitylation of polymerase II in a transcription-dependent manner (87). Although ubiquitylation- mediated polymerase degradation is required for DNA damage repair in yeast (88),the role of this process in human cells is unclear. Recent experiments both in cell extracts and living cells suggest that polyubiquitylation of polymerase II following cisplatin treatment can occur through Lys-6, Lys-48, Lys-63, and possibly other lysines of ubiquitin (82,89). Ubiquitylation may trigger non-degradative signals or affect the properties of stalled polymerase in addition to its degradative roles (90). RNA polymerase II degradation is prevented by the proteosomal inhibitor MG132 with a subsequent increase in the relative amount of ubiquitylated polymerase. Fractionation of polymerase II from cells co-treated with MG132 and cisplatin indicates that this additional ubiquitylated polymerase is mostly unbound or only loosely associated with chromatin (82). Only a fraction of ubiquitylated polymerase II dissociates from damage sites and is destroyed rapidly by proteosomes (Figure 1.3). The effects of platinum adduct on chromatin: nucleosome structure and modification. In a eukaryotic nucleus, DNA is not bare and rather incorporated into nucleosomal fiber, with each nucleosome comprising of a core histone octamer, to form chromatin. 33 Alteration of chromatin properties significantly affects various DNA metabolic processes, such as replication, transcription, and repair. It must be appreciated that platinum drugs modify cellular DNA in chromatin in vivo, and therefore platinum adducts on chromatin will be processed differently from those in free DNA. For example, nucleotide excision repair (NER) of nucleosomal DNA containing a sitespecific platinum lesion is significantly less efficient than that of free DNA containing the same platinum lesion in cell extracts (91,92). Furthermore, the repair efficiency of damaged nucleosomes alters depending on the post-translational modification of histone proteins. The effects of chromatin structure on the reactivity of platinum drugs to DNA have been studied by using reconstituted chromatin as well as various human cell lines (93). The general conclusion is that the linker DNA of chromatin is the preferential target for platinum drugs (94-96), although the effect is diminished at high drug concentrations (96). Overall increase in cisplatin-adduct formation is observed in human cancer cell lines when the cells were treated with arginine butyrate, which inhibits histone deacetylases, affords hyperacetylation of histone proteins, and therefore allows chromatin unfolding (97). Platinum drugs clearly bind more favorably to an open form of chromatin. Structural changes of chromatin by transcription activation (98) or protein binding (99) also modulate cisplatin binding to DNA in human cells. The influence of cisplatin modification on chromatin has also been investigated both in vivo and in vitro. In early studies, chicken erythrocyte nuclei and nucleosomal core particles are treated with cisplatin and the obtained chromatin or nucleosomes are digested by microccocal nuclease (100) and DNase I (101). Digestion profiles indicate that cisplatin binding does not significantly alter the DNA structure of nucleosomal core particle but rather affects the higher order structure of chromatin. This finding is 34 supported by later observation that chromatin remodeling and transcription factor binding are severely impaired by cisplatin modification (71), possibly due to altered chromatin structure. Cisplatin treatment in HeLa cells induced post-translational modification of histones H3 (phosphorylation) and H4 (hyperacetylation) (102), modifications known to modulate chromatin structure. It is unclear at this point if these modifications were direct cellular responses to cisplatin binding to chromatin or indirect results from down-stream cellular pathways following cisplatin treatment. Recently, hydroxyl-radical footprinting was employed for structural analysis of a nucleosome containing a site-specific cisplatin intrastrand cross-link. Cisplatin modification not only changes the rotational phase of DNA wrapping around the histone octamer but also increases the phasing power (103). Enhanced phasing of the nucleosome by cisplatin lesions may explain drug's effect on the higher order structure of chromatin (Figure 1.4). REPAIR OF PLATINUM-DAMAGED DNA Following platinum-induced DNA damage, cellular repair systems immediately act on damage and continuously function until the fate of drug-treated cells is decided. Knowledge of the repair mechanism of platinum-damaged DNA provides essential clues to understand cellular responses to platinum-based anticancer drugs. Nucleotide excision repair. NER is a primary process for repair of platinum-damaged DNA. Bacterial and mammalian cells deficient in NER are more sensitive to platinum drugs (9,104). For example, xeroderma pigmentosum (XP) cell lines, lacking one of the components of the NER process, have increased sensitivity to cisplatin treatment and cell extracts obtained from these cell lines exhibit no repair activity to cisplatin-induced 35 DNA damage (105,106). Cisplatin-resistant tumor cell lines show higher levels of the genes producing NER proteins such as XPC, XPA, and ERCC1 (107), with a concomitant higher repair activity of their cell extracts (108), compared to their wild types. Moreover, enhanced expression of XPC and ERCC1 mRNA is observed in ovarian cancer tissues obtained from patients clinically resistant to platinum compound (109). It is suggested that the exceptional sensitivity of testicular tumors to cisplatin is credited to lower levels of several repair proteins, such as XPA, ERCC1, and XPF, in these cells (110,111). Recently, the enhanced sensitivity to cisplatin of human cancer cells is reported when ERCC1 is suppressed by small interfering RNA (siRNA) (112,113). The molecular mechanism of NER to remove platinum adducts from DNA has been extensively studied (Figure 1.5) (114). During the early stage of NER, platinum lesions are recognized by different mechanisms for two sub-pathways of NER, transcription-coupled repair (TCR) and global genomic repair (GGR). Stalled RNA polymerase II acts as a damage recognition system to initiate TCR as discussed above (77). Cockayne syndrome (CS) proteins, CSA and CSB,are believed to participate in this process although the exact roles of the proteins are unknown (115). For GGR, damage recognition is initiated by XPC-HR23B (116,117). After the initial recognition of DNA damage, TCR and GGR are thought to follow the same events since subsequent NER proteins are required for both GGR and TCR except XPC-HR23B.TFIIH, XPA, and RPA are the next set of proteins to join the damaged DNA. Although the exact binding order of these proteins is controversial, proteins may be cooperatively recruited to the damage site (117,118). In a subsequent step, XPB and XPD helicases, components of TFIIH, unwind the DNA in a process that requires ATP. XPC-HR23B is released when endonuclease XPG binds to this unfolded DNA. Another structure-specific endonuclease XPF-ERCC1 is finally recruited to the NER complex and dual incision 36 occurs to remove platinated oligonucleotides, which are 24-32 nucleotides in length. Excised oligonucleotides, containing a platinum lesion, and dual incision factors are released from DNA. RPA, however, remains associated with the incised DNA and possibly recruits DNA resynthesis factors such as PCNA and replication factor C (RFC) to fill the gap (Figure 1.5) (117). Recently, multiple studies investigated dynamic behaviors of several NER factors such as XPF-ERCC1 (119), TFIIH (120), and RPA and PCNA (121) in living cells. The data consistently indicate that each component of NER diffuses freely and participates in repair processes randomly instead of existing as a repair holo-complex. Most noticeably, dynamic targeting of RPA and PCNA to sites of cisplatin DNA damage is examined in Rat-1 and U20S cells expressing GFP fused to these proteins (121). Cisplatin treatment readily induces the relocalization of PCNA and RPA into discrete foci, while platinum DNA lesions are relatively dispersed throughout the nucleus. PCNA and RPA levels recruited to repair foci are proportional to the platinum adducts level. Proteins at repair foci are highly immobile and turned over only on the order of minutes. The repair of different DNA adducts generated by cisplatin has been investigated in cell-free extracts as well as reconstituted NER systems. In vitro studies with a repair excision or repair synthesis assay revealed that cisplatin 1,3-(GpNpG) intrastrand crosslinks are more efficiently repaired by NER than 1,2 intrastrand cross-links (122,123).The cisplatin interstrand cross-link, however, is not repaired in the same fashion. Other platinum compounds have also been tested for NER of their DNA lesions, which are different from corresponding cisplatin lesions. Intrastrand DNA adducts generated by cisplatin, oxaliplatin, and JM216 (Figure 1.1) are similarly repaired in vitro by NER (124), suggesting that the carrier ligand does not affect the repair efficiency. Although 37 monofunctional adducts of cisplatin and [Pt(dien)Cl]+ are not substrates of NER, several clinically active trans-compounds such as trans-[PtCl2 (NH3 )(thioazole)] (125) and trans- [PtCl2(iminoether)2] (126) form monofunctional adducts, which are successfully removed by the NER system. Monofunctional adducts of these compounds produce a local conformational distortion at the site of DNA damage similar to cisplatin intrastrand cross-links. The efficient repair of DNA adducts generated by a trinulcear platinum complex has also reported (127). Mismatch repair. Numerous studies indicate that the mismatch repair (MMR) process closely correlates with cisplatin resistance (128). Cisplatin-resistant cell lines, which possess the property inherently or acquire it after drug treatment, are often defective in MMR (129,130). Cancer or mouse model cell lines deficient in MMR are several times more resistant to cisplatin than corresponding MMR proficient cells (58). On the other hand, no clear correlation of MMR deficiency with cisplatin resistance was reported in multiple studies (16). The MMR process is likely only one of the pathways linked to cisplatin action, and the influence of MMR on platinum cytotoxicity will vary for every experimental condition. The MMR system eliminates base-base mismatches, and deletion and insertion mutations (131). In eukaryotic cells, hMutScc (MSH2-MSH6 heterodimer) initiates MMR by binding to single mismatches and small insertion/deletion loops, and hMutS3 (MSH3-MSH6 heterodimer) starts MMR through recognition of insertion/ deletion loops of different sizes. Following damage recognition by hMutSa, hMutLc (MLH1-PMS2 heterodimer) and PCNA are recruited to the site of DNA mismatch to proceed the repair. Several exonucleases and helicases, the 38 replication machinery, and DNA ligase I are subsequently recruited to degrade the error-containing strand and fill the gap. MMR proteins reportedly contribute to the cytotoxicity of cisplatin by two distinct pathways (132). First, as a repair process, MMR proteins actively repair the newly synthesized DNA opposite platinum adducts, which is generated from translesion synthesis (TLS) past the platinum lesion as mentioned above. The process can cause a futile repair of cisplatin damage, which ultimately leads to cell death (58). Defects in MMR proteins not only increased cisplatin resistance but also enhanced replicative bypasses of cisplatin adducts (58). MMR proteins are proposed to associate with the replication machinery and to recognize mismatches efficiently on platinumdamaged DNA (131). Binding of MMR proteins to cisplatin-damaged DNA also appears to initiate cellular signals, which lead to programmed cell death (apoptosis) (133). These cellular pathways triggered by MMR proteins are independent from the repair process since certain mutations in hMutS homologs cause mismatch repair deficiencies but do not interfere with the signaling functions of MMR proteins (134,135). Direct interactions of MMR proteins, especially MutS proteins, with cisplatin DNA adducts were studied in vitro. Bacterial MMR protein MutS (136) and its eukaryotic homologues hMutSc (137) and hMSH2 (a component of hMutSa heterodimer) (138) specifically bind to the major cisplatin adduct, a 1,2 intrastrand cross-link. Interestingly, hMSH2 and MutS preferentially recognize the cisplatinmodified DNA over oxaliplatin-modified DNA. Defects in MMR do not affect resistance of oxaliplatin (58), suggesting that the interaction of MMR protein with DNA adducts is important to mediate MMR functions in response to DNA damage. In a recent study, binding properties of MutS (139) and hMutSa (140) to duplex DNA containing cisplatin 39 compound lesions, which possess various mismatches opposite a cisplatin 1,2-(GpG) intrastrand cross-link, were investigated. Cisplatin compound lesions, formed by misincorporation of a base opposite the site of platinum adducts, are better substrates for MutS binding, the affinities of which are changed by the nature of the mismatches. DNA recombination. The roles of recombinational repair for protecting cells from cisplatin treatment have been observed in E. coli (15,141).Many recombination-deficient strains show enhanced sensitivity to cisplatin compared to wild type cells. Recombinational repair is independent from NER since cells containing double mutations in both NER and recombination proteins are more sensitive to cisplatin than cells with either mutation (141). Spontaneous and cisplatin-induced recombination are also observed in E. coli (142). Impaired recombination DNA repair in yeast (143) and prostate cancer cells (144) enhances sensitivity to cisplatin. In mammalian cells, the disruption of homologous recombination repair (HR) increases cisplatin sensitivity, whereas the knockout of the nonhomologous endjoining (NHEJ) does not affect cell's sensitivity to the drug (145). Presently, how recombinational repair proteins specifically encounter platinum adducts is unclear. It was recently proved that collapsed replication forks by strong obstacles on DNA including possibly platinum adducts, recruit recombination proteins to restore the process (146). PROTEINS BINDING TO PLATINUM-DAMAGED DNA Platinum modification distorts the structure of duplex DNA in a distinct way. A variety of cellular proteins specifically recognize this unique form of DNA structure. These proteins include those involved in repair processes, proteins containing HMG domains, and many others. The interaction of the proteins to platinum-damaged DNA 40 plays a key role in early cellular responses to platinum drugs. Continuous efforts have been made to identify such proteins and characterize their interaction with cisplatin adducts. Repair proteins. Damage recognition proteins in various cellular DNA repair processes are reported to bind to cisplatin-damaged DNA. Their general roles in cisplatin action are evident as discussed above, with loss of their functions generally leading to the enhanced sensitivity of cells to the drug. Some repair proteins, however, possess distinct properties to initiate various signaling pathways. NER proteins.Proteins that initiate the NER process are clear candidates to interact with cisplatin DNA adducts. Numerous studies indicate that XPC-hHR23B, XPA, RPA, and TFIIH recognize platinum adducts cooperatively at an early stage of NER (117,147). Among these, XPC-hHR23B, XPA, and RPA all are reported to bind specifically to duplex DNA containing a cisplatin intrastrand cross-link (11). Moreover, these proteins interact with each other, which affects additionally their binding to cisplatin adducts. XPC-hHR23B, a human homolog of yeast Rad4 and Rad23 proteins, shows the highest binding affinity (Kd = -3 nM) to cisplatin 1,3-intrastrand adducts (148). XPC physically interacts with XPA, but the interaction does not contribute to the stability of the XPC-platinated DNA complex. The XPC-XPAinteraction appears to be inhibited by the presence of platinated DNA (118).The XPA protein consists of 273 amino acids (-31 kDa) and contains a zinc finger motif. Although XPA is clearly involved in the NER damage recognition process, it has the lowest binding affinity (-2 tM under physiological conditions) to cisplatin-damaged DNA (149,150).XPA, however, interacts with RPA and the XPA-RPA complex displays a greater binding affinity to duplex 41 cisplatin-damaged DNA than either XPA or RPA alone (151). XPA modulates RPADNA interaction by enhancing the stability of the ternary complex and inhibiting strand separation of the target DNA. RPA is a heterotrimeric protein consisting of 70, 34, and 14 kDa subunits, and an essential component of DNA repair, replication, and homologous recombination. The protein was identified as one of the cisplatin-damaged DNA recognition proteins from the fractionation experiment of human cell extracts by using cisplatin-DNA affinity chromatography (152). RPA specifically recognizes duplex cisplatin-damaged DNA (Kd = 25-79 nM) with about 4-15 fold preference over undamaged DNA, but its binding to single-stranded DNA is also very strong with Kd values in the sub-nanomolar range (151,153).It is proposed that, upon binding to cisplatin-modified DNA, RPA denatures the duplex DNA in the vicinity of the lesion and binds to single-stranded DNA opposite the lesion (154). RPA binds to DNA containing a cisplatin 1,3-intrastrand cross-link 1.52 fold better than to DNA containing a 1,2-intrastrand cross-link, possibly due to low thermal stability of 1,3-adduct compared to that of 1,2-adduct. As mentioned above, XPA enhances RPA binding to platinated DNA (Kd=~0.5 nM), but does not affect RPA binding to single-stranded DNA (151). The p34 subunit of RPA becomes phosphorylated in response to DNA damage in vivo as well as in vitro (155). RPA hyperphosphorylation inhibits its duplex DNA binding but this form of the protein still possesses its binding specificity to platinated DNA (153). XPE-deficient cells display the mildest disorder among XP variants, and these cells still hold 40 to 60% of the repair capacity of normal cells (11). An early study demonstrated that protein extracts of XPE-deficient cells lack a nuclear factor that binds specifically cispaltin-damaged DNA (156).This nuclear factor, called XPE binding factor or UV-damage recognition protein (UV-DRB),is a complex with two subunits of 127 42 and 48 kDa, and recognizes a broad range of DNA damage (157), with its role in damage repair unknown. The protein is induced by cisplatin treatment (158), and cisplatin-resistant cells express increased levels of XPE binding factor (159). Mismatch repairproteins.Damage recognition proteins in MMR, hMutSa (MSH2-MSH6) and bacterial MutS, are reported to bind to cisplatin-modified DNA. The hMutSa heterodimer consists of the MutS homologue hMSH2 and hMSH6 (GTBP/pl60). Purified hMSH2 protein specifically recognizes DNA globally modified by cisplatin (Kd = 67 nM) or [Pt(en)2C12], but not to DNA modified by transplatin and [Pt(dien)Cl]* (138). The protein also binds selectively to 100-bp duplex DNA containing a site-specific cisplatin 1,2-d(GpG)intrastrand cross-link. Human MutSa binds to cisplatin 1,2-d(GpG) (Kd= -25 nM) but not transplatin 1,3-d(GpTpG) adducts (160). hMutSa shows a higher binding affinity to mismatched DNA duplexes than to cisplatin adducts (137). As discussed above, cisplatin compound lesions, such as DNA with a CT sequence opposite a cisplatin 1,2-d(GpG) cross-link site (Pt-GG/CT), are the best binding substrate for hMutSa (140). The interaction of bacterial MutS to platinum adducts was also investigated (136,139). The protein preferentially recognizes globally cisplatinmodified DNA (Kd = -57 nM) over oxaliplatin-modified DNA (Kd = -120 nM) (136). A recent study demonstrated that MutS binds to 24-bp duplex DNA containing a cisplatin 1,2-d(GpG) adduct (Kd= 36 nM) with only 1.5 fold specificity over undamaged DNA, and shows no specific binding to other cisplatin lesions such as 1,2-d(ApG), 1,3d(GpCpG), and interstrand cross-links (139). Similar to hMutSa, MutS strongly binds to cisplatin compound lesions, displaying almost 86-fold better binding affinity to PtGG / CT site than to Pt-GG / CC site. 43 DNA-PK. The DNA-dependent protein kinase (DNA-PK) participates in cellular DNA repair such as DNA double-strand break (DSB)restoration. Recently it has become clear that the protein also plays a central role in various stress signaling pathways (161). DNA-PK is a heterotrimeric complex comprised of a large catalytic subunit (DNA-PKcs) and a Ku70/Ku80 regulatory component with DNA binding properties. Multiple studies reported the involvement of DNA-PK in cisplatin action. DNA-PK mutant cell lines exhibit 3-4 fold increased sensitivity to cisplatin than their parental cell lines, partially due to reduced NER in mutant cells (162). Cisplatin-resistant cells overexpress a Ku80 subunit, with an increased Ku-binding activity of their extracts to DNA ends (163). In a recent study, however, suppression of Ku70 does not affect the sensitivity of cells to cisplatin (164). In addition, cells lacking Ku80 or DNA-PKcs are more resistant to cisplatin than wild type cells but only when the cells are at high density prior to drug treatment (165). The authors suggested that the death signal, initiated in the damaged cell by the kinase activity of DNA-PK complex, is passed to nearby cells by inter-cell communication via gap junctions. DNA-PK binds to globally cisplatin-modified DNA, with the K80 subunit responsible for the interaction (166). Unlike undamaged DNA, which activates the kinase activity of DNA-PK through binding of Ku proteins to DNA ends (161), cisplatin-damaged DNA fails to activate DNA-PK. Ku80 also strongly interacts with DNA containing a cisplatin 1,2-d(GpG) adduct with a Kd value of 0.11 nM, which is only less than 2 fold weaker than to DNA ends (167). Cisplatin DNA adducts appear to inhibit translocation of Ku proteins along DNA, resulting in a decreased association of DNA-PKcs to Ku-DNA complex and therefore a decreased kinase activity (168). The position of the cisplatin adduct and sequence of the duplex DNA affect this inhibition of 44 DNA-PK activity. It was recently reported that DSB nonhomologous endjoining, which requires DNA-PK, is also inhibited by cisplatin-damaged DNA in cell extracts (169). Several other proteins involved in various DNA repair processes are reported to bind to cisplatin-damaged DNA. Yeast photolyase binds to globally cisplatin-modified DNA (170) and E. coli photolyase recognizes duplex DNA containing a cisplatin 1,2-d(GpG) lesion (Kd= 50 nM) (171). Although photolyase appears to make cells more resistant to cisplatin (170,171), the mechanism of this resistance is unclear. T4 endonuclease VII cleaves various branched DNA such as four-way junctions. The enzyme also recognizes and precisely cleaves duplex DNA containing cisplatin 1,2-d(GpG) and 1,2-d(ApG) adducts (172), and interstrand cross-links formed by both cisplatin and transplatin (173). Finally, a recent study reported that human 3-methyladenine DNA glycosylase (AAG), a damage recognition protein of base excision repair, selectively binds to various cisplatin adducts (174). The repair enzyme AAG recognizes 1,2-d(GpG), 1,2d(ApG), and 1,3-d(GpTpG) adducts with Kd values of 115 nM, 71 nM, and 144 nM, respectively. Cisplatin adducts inhibit the AAG repair on 1,N6-ethenoadenine, a wellknown substrate of AAG, possibly by hijacking the enzyme away from the repair process. HMG domain proteins. High mobility group (HMG) domain proteins, particularly HMGB1 protein, have long been connected to cisplatin since the discovery of HMG box binding to cisplatinmodified DNA. Our knowledge of the nature of HMG box interaction with platinated DNA has been greatly improved in recent years. Further investigation, however, is 45 necessary to completely understand the precise effects of HMG proteins on cisplatin anticancer activity. HMGB1. High mobility group (HMG) protein 1 (HMGB1) is one of the early proteins discovered to bind cisplatin-modified DNA (175). HMGB1 is an abundant (106 copies in a cell) and a highly conserved non-histone chromosomal protein (176). This nonsequence specific DNA binding protein regulates numerous nuclear functions including transcription, replication, recombination, and general chromatin remodeling, as an architectural facilitator by assisting the assembly of nucleoprotein complexes (177). HMGB1 preferentially binds to DNA with bent or distorted structures and also physically interacts with various cellular proteins such as p53, RAG1/2, TBP, MutSa and steroid hormone receptors. In one example, HMBG1 binds to MutSa and directs mismatch repair steps prior to the excision of mispaired nucleotides (178). For the past several years, HMGB1 has also been known as an extracellular mediator, performing significant roles in inflammation, differentiation, migration, tumor metastasis and immune response (179). Recently, photobleaching (180) and cross-linking (181) experiments revealed that HMGB1 is an extremely mobile protein in the nucleus and binds to DNA for less than a second. Taken together with high abundance of HMGB1, the protein might have a high chance to encounter platinum adducts and be involved in drug action. The discussed properties of HMGB1 suggest the likely involvement of the protein in cisplatin action, but also make it difficult to expect which aspects of the HMGB1 functions will be mainly engaged in drug action. Not surprisingly, the correlation of HMGB1 levels in cells and cisplatin sensitivity has been controversial. 46 Numerous studies demonstrated that HMGB1 inhibits in vitro NER of cisplatin adducts possibly by binding to and shielding the damage site from repair proteins (122,123). Consistent with these results, an increased protein level of HMGB1 by hormone treatment sensitizes breast cancer cells to cisplatin (182). Moreover, additional expression of HMGB2, a protein over 85% identical to HMGB1, in human lung cancer cells enhanced cisplatin sensitivity more than 3 fold (183). Conversely, HMGB1 is overexpressed in various cisplatin-resistant cell lines (184), and also linked as a proapoptotic signaling protein (185). Mouse embryonic native and its HMGB1 knockout cell lines show no significant differences in their cisplatin sensitivity (186). Recently, RNA interference (RNAi) was employed to silence HMGB1 in three different cell lines, in which the effect of HMGB1 knock-down on cell's cisplatin sensitivity varies in each cell line (187). It is evident that the impact of HMGB1 on cisplatin action is dependent on the cell type and the experimental method used to change the protein level. The effects of foreign HMGB1 on the cytotoxicity of platinum drugs were recently examined (188). Exogenously added recombinant HMGB1 enters HeLa cells and accumulates in the nucleus as well as cytoplasm. Pretreatment of this recombinant protein enhanced cisplatin sensitivity of HeLa and MCF-7 cells by 2 fold and 3 fold, respectively (188), with the sensitization possibly caused by repair shielding of inserted HMGB1. Interestingly, HMGB1 pretreatment also sensitizes HeLa cells but not MCF-7 cells to transplatin through JNK-related signal transductions. This work further demonstrates that HMGB1, as a DNA binding protein as well as a cytokine, have different effects on platinum action depending on the type of cell and the platinum compound. HMGB1, a 30 kDa protein of 215 amino acids, comprises two HMG box domains A and B, and an acidic C-terminal tail. Each HMG domain and full-length HMGB1 selectively bind to cisplatin-modified DNA (189,190). Despite the sequential and 47 structural similarity of the two HMG box domains of HMGB1, domain A interacts more strongly with various cisplatin DNA adducts than domain B (190,191). The sequence context of damaged DNA modulates binding affinity of each domain to cisplatin adducts, such that the Kdvalue of domain A binding to a 15-bp duplex DNA containing a cisplatin 1,2-d(GpG) adduct varies from 1.6 nM to 517 nM depending on the flanking sequence (190). Stopped-flow analysis of domain interaction to cisplatin-modified DNA shows a very fast association (2-4 x 108 M-s-1) and dissociation (70-200 s-1) of protein- DNA complex (192). The crystal structure of domain A bound to a 16-bp DNA duplex containing a cisplatin 1,2-d(GpG) cross-link was determined (193). Structural properties of the HMG box binding to target DNA were reviewed elsewhere (9,42). HMGB1 fulllength protein recognizes cisplatin 1,2 intrastrand (Kd= 0.3 - 370 nM) (189,194)and also interstrand cross-links (195), and the interaction is not affected by sequence context (194). HMGB1 and its didomain protein lacking the acidic tail bind to cisplatin-modified DNA, primarily through domain A with the rest of the protein available for other interactions. The acidic tail of HMGB1 is responsible for the HMGB1 interaction with the TATA box binding protein (TBP) (196). In addition, the acidic tail also appears to interact with histone H3 N-terminal tail and mediate transcription stimulation (197). Enhanced binding of HMGB1 to cisplatin-modified DNA by protein interaction with p53 is reported (198). HMGB1 binding to cisplatin-modified DNA can also be modulated by post-translational modification of the protein. Lysine 2 of HMGB1 is acetylated by histone acetyltransferase CBP (199) and the modified form of the protein shows significantly enhanced binding to cisplatin adducts (200). HMGB1 binding to DNA modified by various cisplatin analogs reveals strong effects of spectator ligands for these protein-DNA interactions. 48 SSRP1. The structure-specific recognition protein SSRP1 was discovered during early searches for the proteins that specifically bind to cisplatin-modified DNA by expression screening of human cDNA clones (201).SSRP1, an 81 kDa protein, forms a heterodimer with Spt16/Cdc68 and the resulting complex FACT (Facilitates Chromatin Transcription) is a chromatin modulator, which mediates transcription, replication, and repair through reconfiguring the nucleosome (202). SSRP1contains a HMG box domain which reflects the protein's binding property to cispaltin-modified DNA. The isolated HMG domain of SSRP1 and the FACT complex selectively recognize cisplatin adducts but SSRP1alone fails to bind this damaged DNA (203). Although the direct evidence of SSRP1 involvement in cisplatin action is not reported, there have been several indications that FACT works in cellular DNA repair processes (204,205). Many other proteins containing one or more HMG domains bind to cisplatinmodified DNA. The proteins include yeast HMG-domain proteins Ixrl (Kd = 250 nM) (206), cmbl (207), and NHP6A (Kd = 0.1 nM) (208), mtTFA (mitochondrial transcription factor A; Kd = 100 nM), LEF-1 (lymphoid enhancer binding protein; Kd = (209), SRY (sex-determining -100 nM) factor; Kd = 120 nM) (210), tsHMG (testis-specific HMG protein; Kd = 24 nM) (211), HMG-D (drosophila homologue of HMGB1; Kd = 200 nM) (212), and hUBF (ribosomal RNA transcription factor; Kd = 60 pM) (213). Similar to human cells, HMG-domain proteins are also closely connected to cisplatin cytotoxicity in yeast. Inactivation of the Ixrl gene desensitizes the yeast strain to cisplatin with a decreased level of cisplatin-DNA adducts (214). On the other hand, nhp6a/b (208) and cmbl (207) mutant cells are more sensitive to cisplatin than their parental cells and cisplatin treatment induces cmbl gene expression (215). In HeLa cells, expression of testis-specific HMG protein tsHMG, which has a higher binding affinity to cispaltin adducts than HMGB1, enhances cisplatin cytotoxicity (216). Finally, in an in vitro 49 transcription assay with RNA polymerase I, cisplatin-damaged DNA inhibited rRNA synthesis by sequestering an essential transcription factor hUBF, which contains six HMG domains and shows the strongest binding affinity to cisplatin adducts, from its natural binding site (217). Other Cellular Proteins. In addition to repair and HMG proteins, many other cellular proteins have been reported to preferentially recognize cisplatin-modified DNA. Since these proteins are ,essential for various cell functions, their interactions with cisplatin adducts are often thought to contribute to cisplatin action. More importantly, in many cases, proteins are ][linkedto other cisplatin damage-recognition proteins through physical or functional communication. TBP. The TATA-binding protein (TBP) is required for transcription initiation of all three eukaryotic RNA polymerases. The protein recognizes a TATA box of the promoter and recruits transcription initiation factors. TBP binds at a minor groove and bends the duplex DNA toward the major groove (218), with the structure of the resulting DNA similar to that of cisplatin-modified DNA. In vitro transcription is inhibited by the presence of cisplatin-damaged DNA, which directly interacts with TBP and sequesters the protein from the TATA box (219). Moreover, microinjection of additional TBP restores inhibition of RNA synthesis in human fibroblasts. Similar to HMGB1, TBP preferentially binds to platinum 1,2-d(GpG) adducts over 1,3-d(GpNpG) adducts (220). Interestingly, HMGB1 binding increases the binding affinity of TBP to TATA box by 20 fold (196), indicating the strong possibility of HMGB1/TBP complex interaction with cisplatin-modified DNA. TBP binding to cisplatin adducts is comparable to its binding 50 to TATA boxes with similar binding affinity and kinetics, which is characterized by relatively slow on and off rates (221). p53. The tumor suppressor protein p53 is found to be most commonly mutated in known human tumors. The p53 protein regulates DNA repair, cell-cycle arrest, and apoptosis through modulation of other genes and their products including those involved in transcription, DNA repair, and many signaling proteins (222). Pathways related to p53 participate in transduction of DNA-damage signals upon cisplatin treatment (16). As recently determined in 60 cell lines, the expression of p53 is positively correlated to the sensitivity to four platinum compounds, which are cisplatin, carboplatin, oxaliplatin, and tetraplatin (223). Numerous reports support this correlation that designed expression of p53 enhances cisplatin sensitivity in p53deficient cancer cells (224,225), and accumulation of p53 also sensitizes cells to the drug (226). In addition, p53 mutants are detected in cisplatin-resistant ovarian carcinoma cells (227). Several studies, however, demonstrated different modulation of cisplatin cytotoxicity by p53. The p53-mediated sensitization to cisplatin is reversed by altering cell growth conditions (228), and p53 expression enhances cisplatin cytotoxicity only in HeLa cells but not in cisplatin-resistant HeLa cells (229). In other examples, p5 3 deficient and -proficient teratocarcinoma cells show the same cisplatin sensitivity (230), and only one of two curable ovarian cancer cell lines displays p53-dependent response upon cisplatin treatment (231).Increased cisplatin cytotoxicity by the loss or abrogation of p53 function is also reported (232). The p53 protein is a DNA binding protein of 393 amino acids, containing two DNA binding domains. The core domain of p53 sequence specifically binds to DNA, and the C-terminal DNA binding domain is believed to recognize various damaged DNA. Under normal conditions, there is a low level of a latent form of p53, which is 51 induced, activated, and stabilized under stress condition such as DNA damage (222). Activation of p53 occurs through post-translational modification, mainly phosphorylation, and this active p53 binds the target DNA sequence specifically. An early study demonstrated that cisplatin treatment to human ovarian cancer cells induces a latent form of p53, and this induced p53 protein lacks sequence-specific DNA binding ability but displays a strong affinity for cisplatin-modified DNA (233). Cisplatin-induced phosphorylation occurs at serine 20 or 15 of the protein (234). Both DNA binding sites are required for p53-binding to platinated DNA (235). Purified active form of p53 recognizes the duplex DNA containing a cisplatin 1,2-d(GpG) adduct (Kd = 150 nM) but not those with 1,3-d(GpTpG), interstrand, and monofunctional adducts (236). Interestingly, the binding affinity of cisplatin-modified DNA to the latent form of p53 is considerably higher than to the active form of p5 3 (237). Both latent and active p53, however, do not bind to DNA modified by the trinuclear platinum drug, 13BR3464(238). As discussed briefly, p53 interacts with various cisplatin damage recognition proteins (XPC,RPA, YB-1,HMGB1, and mtTFA), and significantly enhances binding affinities of HMGB1 (198) and also mtTFA (239) to cisplatin-modified DNA through physical interaction. PARP-1. Poly(ADP-ribose) polymerase 1 (PARP-1) is a large (1014 amino acids) nuclear enzyme that utilizes NAD+ as a substrate for the synthesis and attachment of poly(ADPribose) polymers to target proteins as well as to itself in response to DNA damage (240). Severe DNA damage appears to cause overactivation of PARP-1, which leads to the depletion of NAD+ and ATP, and ultimately to necrotic cell death. Evidence correlating PARP-1 and cisplatin action are very limited at this point. Cisplatin treatment, however, increased overall poly(ADP-ribosyl)ation in 0-342 rat ovarian tumor cells and CV-1 monkey cells (241), where PARP-1 is a main contributor to the modification (240). 52 Moreover, PARP-1 inhibitors sensitize various human cancer cell lines to cisplatin (242,243).Recently, PARP-1 was identified as one of nuclear proteins that selectively bind to cisplatin 1,2-d(GpG) adducts by using photoaffinity labeling (244).Possible roles of PARP-1 in cisplatin anticancer activity are discussed in a recent review (245). YB-1. The Y-box binding protein YB-1is a transcription factor that binds to the Y-box, an inverted CCAAT box sequence, and is important for signaling DNA damage and cell proliferation. This protein is overexpressed in the nucleus of cisplatin-resistant cell lines (246,247),and suppression of the protein increases cisplatin sensitivity of human cancer cell lines or mouse embryonic stem cells (246,248). The mRNA level of YB-1 is increased about 6 fold in response to cisplatin treatment (249). YB-1 selectively recognizes the DNA duplex containing cisplatin 1,2-d(GpG), 1,2-d(ApG), as well as 1,3-d(GpTpG) adducts and physically interact with PCNA, suggesting possible involvement of the protein in DNA repair (250). YB-1 also interacts with many cellular proteins such as MSH2, DNA polymerase 6, Ku80 (251), and p53 (252),which are key proteins discussed here for their functions in cisplatin action. CONCLUDING REMARKS Platinum-based anticancer drugs such as cisplatin, carboplatin, and oxaliplatin are among the most widely used chemotherapeutic agents. Challenges for researchers in this field have been to minimize side effects of the drugs while maintaining their potency against cancer cells and to extend successful treatment to a wider range of human cancers. The search for novel platinum drugs and better therapeutic strategies demands a deeper understanding of how cells process platinum drugs. Recent progress provides more clues to explain these courses of cellular action including platinum-DNA adduct formation after drug uptake, DNA damage recognition by damage-response 53 proteins, and subsequent signaling pathways, which decide the result of drug treatment. Proteins mediating direct cellular responses to platinum-damaged DNA include those involved in replication, transcription, repair, and chromatin structure, as well as proteins specifically binding to platinum-DNA adducts. Many of these proteins physically and functionally communicate with each other, which will further affect their roles in cisplatin anticancer activity. 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Cell Biol. 34, 87-92 72 Table 1.1 Key human proteins that bind to cisplatin-modified DNA protein function Kd specificity note ref 3 nM nd (118,148) 0.4-2 [tM < 3 fold recognition protein 25-79 nM 4-15 fold hMSH2 MMR: damage recognition protein -67 nM 5 fold hMutSa MMR:damage recognition protein -25 nM >10 fold 0.11 nM nd NER: damage XPC recognition protein NER: damage XPA recognition protein NER: damage RPA DNA-PK: DNA Ku80 binding subunit Non-histone chromatin protein and HMGB1 extracellular 0.3 -370 nM 10-100 fold interact with XPA interact with RPA and XPC interact with XPA (149,150) (151-154) (138) high specificity for compound lesions interact with PARP1 (137,140,160) (166,167) interact with p53, TBP,and MutSa (189,194,195,1 98,200,253) need to form a FACT complex for (203) signaling protein Chromatin modulator >0.3 jiM hUBF rRNA transcription factor 60 pM nd (213,217) tsHMG Testis-specific HMG protein 24 nM 230 fold (211) TBP Transcription initiation factor 0.3 - 10 nM nd interact with p5 3 Tumor suppressor protein -150 nM nd RPA, YB-1, HMGB1, SSRP1 Poly(ADP-ribose) PARP-1 polymerase Y-box binding YB-1 - transcription factor I >50 fold binding HMGB1 (219,220) interact with XPC, (235-238) and mtTFA nd nd interact with DNA- nd nd interact with MSH2, PK Ku80 and p53 (244) (250) 73 0o H3N\ /CI Pt Cl H3 N H3 N H3N/ 0 Pt \0 H3N, I I Pt H 2N I *CI 0 Cisplatin Carboplatin JM216 H2 N\ /CI H3N\ Pt /CI NH Pt CI NH2 NH3 Transplatin Oxaliplatin [Pt(dien)Cl]' Figure 1.1. Cisplatin and related platinum-based anticancer drugs. 74 H A + H3 N\ /CI Pt H3 N Ci L C B 1,2-d(GpG) 1,3-d(GpTpG) 1, , r r1 i . · 9 t* I~~~~~I Figure 1.2. Platinum DNA adducts: formation and structures. (A) Cisplatin is activated by hydrolysis in the cell and the activated form of cisplatin, [Pt(NH3)CI(OH2)] +, binds covalently to the N7 position of purines. The structures of duplex DNA containing (B) cisplatin 1,2-d(GpG) (43) and (C) 1,3-d(GpTpG) intrastrand (44) are generated by PyMol. 75 ....................................... mIDK^IA ... lrr Stalled Pol II Ubiquitin I; - Platinum adduct Release factors (HuF2) Multiple ubiquitin ligases CSB, TFIIS, k~~~~~~~~~~~~. -- )> I . I ~, r I I. Pol II Release II. Backtracking & Re-elongation IV. Non-degradative 4 GGR? ` TCR? Signals? III. Pol II Release & Degradation GGR? Figure 1.3. Schematic representation of transcription inhibition by platinum lesions and consequent outcomes. 76 Cisplatin n Cisplatin adduct * Histone modification: Phosphorylation & acetylation + * Post-translation modification of Histones · Alternation of the rotational phase · Enhancement of the phasing power Inhibitionof cisplatin adducts repair Inhibitionof chromatin remodeling Inhibitionof transcription factor binding Figure 1.4. The effect of platinum damage on chromatin structure and function. 77 Transcription-coupled repair Global genome repair Stalled Pol II m "Mw Pt Pt XPA, RPA, TFIIH RPA XPA XPG, XPF-ERCC1 RPA | XPA rst Dual incisioDNA resynthesis RPA, Figure 1.5. The mechanism of nucleotide excision repair of platinum lesions. 78 Apoptosis | I Signal transduction Fu, ~TMut '5IM hMutS, DNA pol | Ku80,p53 Protein C. ' interaction :I I Poly(ADP-ribosyl)ationenhancement I * DNA-PK function inhibition Directsignaling tilerepair I I Death signal transduction / ! + / I Repair shielding Cisplatin I .K ;_-._L_.,=__ rtK inniDuon1 DNA damage (_" ARP-V MGBI-O - /1) Necrotic cell death I *ATPINAD+ depletion .. P911;3p'j i ~/ Y Death signal transduction Protein 53 I interaction hMutS, TBP, p mo -q Ifi I \ Protein I Cytokine signal transduction \ roteinhijacked interaction Transcription inhibition XPC, RPA, YB-1 HMGB1, mtTFA Figure 1.6. The roles of proteins binding to platinated DNA in cisplatin anticancer action. 79 Chromatin alternation Affectsall directcellular ' responsesto Pt-DNA t Replication Protein-protein interaction Cisplatin DNA damage *4 inhibition / Damage recognition by cellular proteins I Transcription inhibition Cell cycle arrest Direct signaling I~"rr' TCR IMlA UnnI . uarri repair Apoptosis ellsurvival Cell survival Necrosis Cell death Figure 1.7. Direct cellular responses to platinum adducts: overall picture of current understanding. 80 Chapter 2 Kinetic Studies of the TATA-BindingProtein Interaction with CisplatinModified DNA *The work in this chapter has been published in J. Biol.Chem.,2001,276, 43589-43596. 81 Introduction Following the discovery of the anticancer activity of cisplatin, many studies have focused on elucidating its mechanism of action (1,2). The formation of covalent cisplatin-DNA adducts, especially the 1,2-intrastrand d(GpG) cross-link, correlates with the cytotoxicity of the drug (1-3). Attention now focuses on understanding how cells react to the presence of the cisplatin-DNA lesions and, using this information, designing more effective anticancer treatments. Upon cisplatin binding, the DNA duplex is bent and unwound, and the minor groove becomes wide and shallow. These structural changes inhibit essential DNA metabolic processes such as replication and transcription (4-7). The distorted DNA duplex can also interact with a number of cellular proteins (1,2,8,9),an activity postulated to mediate the processing of cisplatin-DNA lesions (13,10-14). The identification and characterization of proteins that bind selectively to cisplatin-damaged DNA has therefore become one of the main thrusts of research in this field. The TATA-binding protein (TBP) is a key component of TFIID, which is required for transcription initiation of all three eukaryotic RNA polymerases (15). As the first step in the process, TBP recognizes a TATA box element located -30-bp upstream from the transcription start site and eventually recruits other transcription factors. Structural analyses of several TBP-TATAbox complexes reveal that TBPbinds at a widened minor groove and bends the duplex DNA toward the major groove by intercalation of two pairs of phenylalanine residues (16-18).Since sequence-specific DNA-binding proteins usually reside in the major groove, the minor groove binding by TBP was initially puzzling. Subsequent studies demonstrated that the flexibility of the TATA box element primarily determines its binding affinity to TBP (19,20).In addition to the specificity of 82 its DNA binding, TBP has distinctive DNA-binding kinetics. The formation of the TBPDNA complex is characterized by relatively slow on and off rates (21). TBP binds selectively to cisplatin-damaged DNA (22). The sequesteration of TBP by cisplatin DNA adducts inhibits transcription in vitro, which could be restored in a reconstituted system by addition of extra TBP (22). Transcription inhibition by exogenously added cisplatin-damaged DNA has also been reported (23).More recently, enhanced binding of TBP to the TATA element containing flanking cisplatin 1,2intrastrand cross-links was demonstrated (24). This series of experiments suggests a possible role for TBP-cisplatin-DNA ternary complexes in mediating the anticancer activity of the drug. Because a variety of proteins interact with cisplatin-DNA adducts (9), many studies have focused on determining their binding affinity (25-30). In order to evaluate the relative importance of the different protein-platinated DNA interactions in the cell, however, detailed kinetic parameters are also required. In spite of the importance of such information, few attempts have been made to examine the kinetics of protein binding to cisplatin-damaged DNA (31-33).The interactions between the two domains (A, B) of HMGB1 with cisplatin-modified DNA were investigated by using stoppedflow fluorescence and fluorescence resonance energy transfer (FRET) methods (31,32). In addition, a stopped-flow kinetic analysis was performed for replication protein A (RPA) binding to cisplatin-damaged DNA (33). In the present study we employed electrophoretic mobility shift assays (EMSAs) to examine the kinetics of TBP binding to cisplatin-damaged DNA and, for comparison purposes, the TATA box. TBP binding to DNA containing a site-specific 1,2-intrastrand d(GpG) cross-link was evaluated by using several platinum-modified DNA probes with various flanking sequences. We also performed an in vitro repair assay in order to 83 examine the effect of TBP in modulating nucleotide excision repair (NER) of platinated DNA. The results provide strong evidence for a biological role of TBP in mediating the cytotoxicity of cisplatin. Experimental Procedures Preparationof Yeast TBP. The C-terminal DNA binding domain of recombinant yeast TBP was provided by Dr. S. M. Cohen in our laboratory and prepared as described previously (24). The concentration of active TBP was determined as reported in the literature (34). Preparation of Oligonucleotides Probes. Table 2.1 lists the 25-base-pair (25-bp) oligonucleotides used in this study together with their abbreviations. The synthesis, platination, and purification of site-specifically platinated single-stranded oligonucleotides were carried out as described previously (26). Platinated top strands, (5'-CCTCTCCTCTCN1 G*G*N2TCTTCTCTCC-3', N = A, T, or C), where the asterisks indicate the formation of Pt-N(7) bonds, were annealed with their complementary bottom strands in 10 mM Tris (pH 7.0), 50 mM NaCl, and 10 mM MgC12, heated to 90 °C, and slowly cooled to 4 C over several hours. The resulting cisplatin-modified duplex probes were purified by ion-exchange HPLC using a Dionex DNApac PA-100 column. These oligonucleotides were desalted by dialysis and concentrated to 5-10 M. The TATAMLP probe (Table 2.1) was prepared by the same method except for the platination step. The TATA element of adenovirus major late promoter, one of the strongest promoters, was used for this study. The purity of the platinated singlestranded DNA was confirmed by analytical HPLC. Atomic absorption spectroscopy combined with UV/vis spectroscopy and electrospray mass spectrometry verified the existence of singly platinated probes (Table 2.2). 84 Electrophoretic Mobility Shift Assay (EMSA). All platinated and TATAMLP duplex probes (10 pmol) were radioactively labeled at their 5'-ends by using 50 iCi of [y32 P]ATP (Dupont/NEN) and 20 U of polynucleotide kinase (New England Biolabs). Labeled probes were separated from small nucleotides by passage through G-25 Sephadex Quickspin columns (Boehringer Mannheim). For each binding reaction, DNA probes (0.5 - 2 nM, ~10,000 cpm) and the indicated concentrations of TBP were mixed and incubated in a buffer solution containing 60 mM KC1,20 mM Tris (pH 7.9), 5.0 mM MgC12, 10 mM DTT, 0.2 mg/mL BSA, and 10% glycerol (24). After incubation at 30 °C for 30 min, binding mixtures were directly loaded onto 6 % native polyacrylamide gels and electrophoresed for 1.5 hr at 150 V in 25 mM Tris (pH 7.9), 190 mM glycine, 1.0 mM EDTA, and 4.0 mM MgC12 running buffer. The gels were dried at 80 C and then exposed to phosphorimager plates for 15 - 20 hr. Quantitative analysis was performed with a Bio-Rad GS-525 Molecular Imager employing Multi-analyst software (Bio-Rad). Kinetic EMSA Analysis. For the measurement of the protein-DNA association constants by kinetic methods, the increase in the amount of complex was monitored at various times after addition of 5 - 10 nM of TBP to 1 nM of the probe. As described previously (35), the raw data were fit to eq 1, which is derived from the bimolecular 1/ [TBP] 0 1n{[probe] 0 / {[probe]o - [complex]}} = kont (1) binding equation under conditions of excess protein (36). The left side of the equation was plotted against time in sec and the ko value for each probe was obtained from the slope of the plot. In the equation, [TBP]Oand [probe]oindicate the initial concentrations of TBP and DNA probe, respectively, and [complex] is the concentration of complex at each time point. 85 In order to determine the dissociation rate constants (koff),-10 nM of TBP and -1 nM of probe were mixed at 30 °C for 30 min to reach the equilibrium state. Dissociation of the protein-DNA complexes was initiated by addition of 200 ng of poly[dI dC] at different time points in order to assure that all dissociation reactions were complete at the same time. The decrease in the amount of complex was followed over a 1- or 2-hr time period. The data were fit to eq 2 to obtain the first-order rate constant for the ln{ [complex] / [complex]o I = -kofft (2) dissociation reaction, where [complex] indicates the concentration of complex at time t and [complex]0 is the complex concentration under the initial binding conditions. The natural logarithm of [complex] divided by [complex]owas plotted versus time and the negative slope of the fit provided the kff value. The dissociation constant, Kd,values were calculated from these results (Kd=kon/koff). CompetitionAssay. Competitor DNA of varying concentrations was mixed with a fixed amount of TBP and radioactively labeled TATAMLP, and the disappearance of the TBP-TATAMLPcomplex was analyzed by EMSA. Competition assays were also used to obtain the Kd values for weak binding probes. In such a competition experiment, two binding reactions proceed simultaneously and follow the relationship in eq 3 (37), where P, C, and Tt represent 0= P(1- ) Kt(1 + Ct)/K + Tt(1- 0) (3) 86 the total concentrations of TBP, competitor, and TATAMLP,respectively. Kt and Kcare the respective dissociation constants of TATAMLP and competitor. At different Ct values, the binding fraction (0) was determined. The Kdvalue can be calculated by using eq 4, where C1 /2 represents the concentration of competitor at 0 Kc 2KtC1/2 2KtC/2 2Pt - Tt - 2Kt 1/2 (37). (4) ExcisionRepairAssay. Whole cell-free extracts from HeLa cells were prepared by a reported method (38) and stored at -80 °C.DNA duplexes 161-bp in length containing a site-specific cisplatin 1,2-d(GpG) or 1,3-d(GpTpG) cross-link with a radiolabeled phosphate located six or seven bases to the 5' side of the cisplatin binding site were prepared as previously described (39,40). The probes contained 3 phosphorothioate residues at their 3' ends to minimize non-specific nuclease degradation. Whole cell extracts were incubated with damaged DNA and excision fragments were resolved by 10% denaturing PAGE. For the repair experiment in the presence of TBP, the protein was pre-incubated with the damaged DNA probe in excision repair buffer for 30 min on ice unless otherwise indicated. The extent of NER was measured by comparing the signal intensity corresponding to the 25-30-bp excised fragment with that of the entire lane. The small amount of DNA degradation due to non-specific nuclease activity was subtracted from the repair signal by using the area corresponding to the 32-37-bpregion as background (41). FootprintingAssay. The DNA duplex, TATAMLP or AGGA (Table 2.1), in which only a single strand was labelled at the 5'-end phosphate, was mixed with TBP in the same buffer used in the EMSA except glycerol and DTT. The binding solutions were incubated on the ice for 30 min before the hydroxyl radical reaction. Cleavage reactions 87 were performed in a solution containing 1 mM of [Fe(EDTA)] 2-, 10 mM of ascorbic acid, and 0.15 % aq. H2 02 . Obtained oligonucleotides were analyzed on a 20% polyacrylamide denaturing gel. Results Kinetics of TBP Binding to the TATA Box and Cisplatin-DamagedDNA. In order to optimize conditions for the EMSA experiments, probes of three different lengths (15, 25, and 35 bp) were investigated. The 15-bp probe showed weak binding affinity to TBP compared to longer probes. In addition, this short oligonucleotide was partially melted under the experimental conditions (data not shown). The 35-bp probe displayed a considerable level of non-specific binding (data not shown). The 25-bp probe was stable under the experimental conditions and did not exhibit any non-specific binding. Therefore, 25-bp probes were used for further studies. EMSA analyses were performed to investigate the kinetics of TBP binding to the TATA box (TATAMLP) and cisplatin-damaged DNA (AGGA, Table 2.1). Figure 2.1A shows EMSA data used to determine the association rate constants for two probes, and the data analysis is presented in Figure 2.1B. The bimolecular binding reactions reach the equilibrium state within 10 min for both probes. As shown in Figure 2.1A, however, the TBP-AGGAcomplex clearly forms more rapidly than the TBP-TATAMLPcomplex. Data collected within 2-3 min were fit to eq 1, yielding konvalues of 1.3 x 105 M-ls-l for 113Pbinding to TATAMLP and 3.0 x 105 M-ls- for the AGGA probe (Figure 2.1B). The kon value for TATAMLP is in good agreement with published values, which fall in the range 1.0 - 3.0 x 105M ls' 1 (21,35,42-44). Following addition of excess of poly[dIdC] to the TBP-DNA complex, decreased amounts of the complex were detected (Figure 2.2). The dissociation reaction data were 88 fit to eq 2. The AGGA and TATAMLP probes dissociate from TBP with half-lives, t/ 2, of 120 min and 190 min, respectively. Although TBP associates with AGGA more rapidly than with TATAMLP,the latter probe has a slightly lower off-rate (Table 2.3). From these konand koffresults, dissociation constants Kd(koff/kon)for each probe were calculated (Table 2.3). Comparison of Kdvalues for AGGA and TATAMLP reveals that cisplatin-damaged DNA has about a 1.5-fold higher binding affinity to TBP compared to that of its natural binding site, the TATA box, under the same binding conditions. The Kd value (0.44 nM) for the TATA box binding to adenovirus major late promoter is consistent with previous reports (21,34,35,42-45). FlankingSequencePreferenceof TBP Binding to Cisplatin-DamagedDNA. Previously we demonstrated that HMG domains display a distinct selectivity for platinated DNA containing different flanking sequences (26,46).Moreover, different flanking sequences can affect the conformational and thermodynamic properties of cisplatin-damaged oligonucleotides (47). Here, we studied the kinetics of TBP binding to cisplatindamaged probes in diverse sequence contexts (Table 2.1). Figure 2.3 shows EMSAs of TBP binding to TATAMLP and nine different cisplatin-damaged probes. Sequence selectivity is clearly manifest for TBPbinding to platinated DNA. Kinetic EMSA experiments were performed to determine konand koff values for each probe. Figure 2.4A indicates fits to EMSA data obtained from the association kinetics for six different cisplatin-damaged probes. All kon values lie between 2.1 x 105 M-'s-1 and 1.2 x 105M'ls- , indicating that most of the cisplatin-damaged DNA complexes have faster association rates than that of the TATA box (Table 2.3). The kff values were also calculated by fitting the dissociation EMSA data to eq 2 (Figure 2.4B). The two more weakly binding probes, TGGC and CGGC, did not allow reliable kinetic data to be obtained. We therefore carried out competition assays to obtain relative Kd values for 89 these two probes. At various concentrations of TGGC or CGGC competitor, the binding fraction of the TBP to TATAMLP was examined by EMSA and quantitated (Figure 2.5). From the curve fitting shown in Figure 2.5B, the concentration of competitor at 0 = 1/2, C1 /2, was calculated. Eq 4 was employed to compute Kd values of 12 nM and 10 nM, respectively, for TGGC and CGGC as competitors. Our results reveal a clear flanking sequence dependence of TBP binding to cisplatin-damaged DNA (Table 2.3). At the N, position, TBP prefers dA rather than dT or dC, and TBP forms more stable complexes when N2 is either dA or dT. Cisplatin-Damaged DNA Sequesters TBP from the TATA Box. A DNase I footprinting assay previously demonstrated reduced binding of TBP to the TATA box following addition of excess cisplatin-damaged DNA (48). In the present study, competition EMSA experiments were performed to study the relative binding affinities of TBP to the TATA box and cisplatin-damaged DNA. TBP and the two DNA probes were mixed and the amounts of free and bound TATA box were analyzed by using radioactively labeled TATA box DNA. Figure 2.6 provides clear evidence that TBP dissociates from the TATA box upon addition of cisplatin-damaged DNA. At 1 nM TBP, more than half of the TBP-TATAMLP complex has dissociated when equivalent amounts (4 nM) of AGGA and TATAMLP are present. Upon addition of a three-fold excess of AGGA, most of the TBP is sequestered from the natural TATAMLP binding site. TBP BlocksNER of the Cisplatin1,2-d(GpG)Cross-Link.Cisplatin-modified DNA is repaired by the NER machinery. When HMG-domain proteins are added to HeLa cellfree extracts or reconstituted NER components, a significant amount of the repair of cisplatin 1,2-d(GpG) intrastrand cross-links is inhibited (39). Repair of cisplatin 1,3d(GpTpG) intrastrand cross-links, which do not bind HMG-domain proteins, was not 90 significantly affected under the same conditions. Since TBP has a high binding affinity for cisplatin 1,2-d(GpG) intrastrandcross-linked DNA, we next examined repair of this adduct by NER using HeLa whole cell-free extracts in the presence of TBP. For this experiment we employed the CGGC probe because its repair signal is larger by a factor of two than that of AGGA (data not shown). Figure 2.7 reveals that repair of the cisplatin 1,2-d(GpG) intrastrand cross-link in CGGC is indeed inhibited by pre-incubation with TBP, whereas a probe containing the cisplatin 1,3-d(GpTpG) intrastrand cross-link is not. In the presence of a great excess of TBP (> 200 M), repair of the cisplatin 1,3-d(GpTpG) cross-link is also shielded to a similar extent as the 1,2d(GpG) cross-link, probably because of non-specific binding (data not shown). TBP Binding Mode to Cisplatin-Damaged DNA. Hydroxyl radical footprinting assays were employed to study TBP interactions with the DNA duplex containing a TATA box or a cisplatin 1,2-d(GpG) lesion. For both probes approximately 12-bp DNA fragments are protected from hydroxyl radical by TBP (Figure 2.8). The TATA box and the cisplatin d(GpG) lesion are located in the middle of the protection region. Discussion TBP Binding to Cisplatin-DamagedDNA. The present study reveals that TBP very slowly associates with and dissociates from cisplatin 1,2-d(GpG) intrastrand cross-links, behavior that is very similar to its binding to the TATA box. Including these results, kinetic parameters for the binding of three proteins, HMGB1-domain proteins, TBP, and RPA, to cisplatin-modified DNA are now available (31-33). Although it is hard to compare the kinetic parameters directly, owing to the different binding conditions and temperatures employed, TBP clearly exhibits very different properties for the binding to cisplatin-damaged DNA. The konvalues reveal that the HMGB1 domains bind to 91 cisplatin-modified DNA near the diffusion limit (kon= 5 x 108M1- s-l), more than 1000fold more rapidly than the association of TBP (kon= 3.0 x 105M1- s1). RPA also has about a 100-fold higher association rate constant (kon = 2.2 x 107 M-1 s) than TBP. If the association rate were the determining factor in protein binding to target lesions in the cell, TBP would be an unlikely participant in the competition for cisplatin-modified DNA. However, HMGB1 domains and RPA also dissociate much more rapidly than the extremely stable TBP-platinated DNA complex. Under relatively similar binding conditions, TBP has more than a 100-fold higher binding affinity for cisplatin-damaged ]DNA than RPA (33). In addition, TBP has the highest specificity for cisplatin-damaged DNA among all the identified proteins (1). Since the Kdfor TBP binding to genomic DNA is 6 x 10-6 M, this protein prefers to bind platinum-damaged DNA by a factor of >3000 compared to undamaged DNA (34). RPA and HMGB1 have ~15- and 100-fold preferences for damaged over undamaged DNA, respectively (25,28,49). Several protein-protein interactions involving TBP, RPA, and HMGB1 occur that can affect the DNA binding kinetics in the cell. XPA, another damage recognition protein, stabilizes the RPA-damaged DNA complex (28,50). Interactions between human TBP and HMGB1 occur by which the latter inhibits transcription (51). Subsequent studies revealed that HMGB1 enhances the binding affinity of TBP to the TATA box by about 20-fold, with the C-terminal acidic domain of HMGB1 and the nonconserved N-terminus (residues 55-95) of human TBP being essential to complex formation (52-54). Since these two highly abundant proteins can bind to cisplatin- damaged DNA, and the interaction between them occurs through their non-DNA binding domains, the binding kinetics of the HMGB1-TBP complex to cisplatin-DNA lesion would be interesting to evaluate. Ultimately, information about the DNA binding 92 kinetics of RPA-XPA and HMGB1-TBP complexes will be useful for assessing likely protein binding priorities in the cell. Flanking Sequence Dependenceof TBP Binding to Cisplatin-DamagedDNA. Kinetic analyses of TBP binding to 10 different probes were performed and the results are summarized in Table 2.3. The trends in the kinetically determined Kd values of the individual probes agree well with the EMSA data in Figure 2.3. Both konand koffvalues generally affect the binding affinities of the probes studied. This binding is unlike that encountered in classical TATA box kinetic studies, in which different TATA boxes have different koffvalues (24,35,44).Specifically, it is the differences in konvalues that mainly determine the relative TBP binding affinities between the three probes AGGA, AGGT, and AGGC. The flanking sequence dependence of TBP binding to cisplatin-damaged DNA reveals a preference for dA rather than dT or dC at the N1 position. For the N 2 position, TBP shows higher binding affinity for dA or dT compared to dC. These flanking sequence preferences for TBP binding are surprisingly similar to those for HMGB1 domain A binding (26,46). The two proteins, HMGB1 domain A and TBP, share no sequence or structural homology, but they display similar selectivities for DNA sequence context of cisplatin-DNA 1,2-intrastrand cross-links. In order to address the reasons for this selectivity, we now consider the probable binding mode of TBP to cisplatin-damaged DNA. TBP binds to the widened minor groove of the TATA box mainly by hydrophobic interactions. Two key features of such binding are intercalation by two pairs of phenylalanine residues and several hydrogen-bonding interactions involving two asparagine residues at the middle of complex (19). The TATA box used in the present study and several of its specific interactions with TBP are shown in Figure 2.9A. 93 In previous work, a DNase I footprinting assay was performed for TBP bound to the TATA element and to cisplatin-damaged DNA (48). TBP protected 12-bp of DNA in a region including the TATA box, as indicated in Figure 2.9A. Similarly, a 4-bp DNA fragment at the 3' side and at least a 6-bp DNA fragment at the 5' side of the cisplatin d(GpG) 1,2-intrastrand cross-link were protected by TBP (Figure 2.9B). We carried out hydroxyl radical footprinting assays, which provide higher resolution footprints of DNA-protein complexes (55,56). Consistent with previous results, the same 12-bp DNA fragments around the cisplatin d(GpG) lesion and the TATA box were protected by TBP (Figures 2.8 and 2.9). Evidently, these footprinting results cannot reveal the exact intercalating positions of TBP bound to cisplatin-damaged DNA, which determine its precise binding mode. Nevertheless, the approximate positioning of the N1 and N2 nucleotides is possible to delineate. The N2 nucleotide is near the intercalating position and the N1 nucleotide is at the hydrogen-bond contact region at the center of complex (Figure 2.9B). The preference of dA or dT at the N2 position for TBP binding suggests that it is the flexibility of base pairing at N2 that determines the binding affinity (19,20). For the N, position, TBP prefers to bind probes containing dA at N, rather than dT or dC. Specific interaction between dA and residues in TBP possibly contribute to this selectivity since N, is near the two-asparagine contact region. More details about the binding mode of TBP-platinated DNA complexes must await an X-ray structure determination. In spite of the similarity in flanking sequences preferences, TBP is much less selective than HMGB1 domain A. The highest affinity probe, AGGA, has only 40-fold lower Kdvalues compared to the weakest (TGGC) probe for the interaction with TBP. In contrast, the binding affinities of HMGB1 domain A vary by several orders of magnitude depending upon the sequence context of the cisplatin-DNA lesion, and the 94 lowest affinity probe has only several-fold higher binding affinity than that of undamaged DNA (26). Since different flanking sequences have the same likelihood of occurring in platinated DNA (57), some cisplatin DNA lesions may not participate at all in interactions with HMGB1 in the cell. A crystal structure of HMGB1 domain A bound to a cisplatin-modified DNA duplex revealed the intercalation of a single phenylalanine residue into the hydrophobic wedge created in the minor groove across from the platinum-induced d(GpG) cross-link, with its binding extending mainly to 3' side of the adduct (58). Since this intercalation interaction is critical for tight binding of HMG1domain A, the flanking sequence significantly affects the binding affinity of the protein to cisplatin-modified DNA (26). In contrast, in the TBP complex, the cisplatin d(GpG) adduct is located between the two intercalation sites, resulting in a much less significant flanking sequence influence on binding affinity. Although the two proteins have different degrees of selectivity for flanking sequence, their preference trends are similar. In both cases, flexibility at the flanking base pairs is a primary contributor to binding selectivity. BiologicalImplications of TBP Binding to Cisplatin-DamagedDNA. In the cell the TBP-TATA interaction is required for the recognition and utilization of eukaryotic promoters in basal transcription. The formation rate and stability of TBP-TATA complexes are major determinants that regulate transcription (43,59,60).Disruption of this important interaction is an attractive candidate for explaining the cytotoxicity of cisplatin. In the present investigation, one of the site-specifically cisplatin-modified probes, AGGA, showed a Kdvalue of 0.3 nM, which indicates 1.5-fold higher binding affinity to TBP than a strong TATA element. Such high binding affinity of cisplatin-damaged DNA for TBP suggests that the drug can interfere with cellular TBP-TATAinteractions. 95 Furthermore, cisplatin lesions with different flanking sequences show similarly high binding affinity to TBP (Table 2.3), as discussed above. Even the most weakly binding probe, TGGC, has sufficient binding affinity (Kd = 12 nM) to disturb the TBP-TATA interaction, because many weak promoters have TATA elements with Kdvalues higher than 12 nM (19,43).In the mammalian genome, approximately 10,000transcribed genes contain the TATA box in their promoters (2,61). Tumor cells isolated from cisplatintreated cancer patients have 10,000-100,000 platinum lesions per genome (2,62,63). These data and our kinetic results suggest that the hijacking of TBP from its natural binding site, the TATA element, might contribute to the cytotoxicity of cisplatin. Nucleotide excision repair (NER) is a major pathway for removing DNA lesions including those of cisplatin (64). Specific interactions between cisplatin-damaged DNA and damage-detection components of NER such as XPA and RPA have been extensively investigated (65-67). In addition, study have demonstrated that diminished repair in tumor cells sensitizes them to cisplatin (68). In addition to the NER components, there has been much interest in the role of other cellular proteins binding to cisplatin-damaged DNA. Previous work revealed that HMGB1 proteins block cisplatin lesions from NER as monitored by a repair assay conducted with whole cell extracts (12,39). In addition, a yeast mutant lacking the HMG-domain protein Ixrl was less sensitive to cisplatin than wild type cells (11). HMGB1 has been implicated as an important protein in the modulation of such repair shielding owing to its abundance in the cell and its relative high binding affinity for cisplatin lesions (1,11,12,25,39).From this work has emerged a strategy for enhancing cisplatin activity in the clinic (13). In the present study, TBP is demonstrated to shield cisplatin-damaged DNA from NER. The repair of the intrastrand 1,2-d(GpG) cross-link is specifically reduced following pre-incubation with TBP, whereas repair of the intrastrand 1,3-d(GpTpG) 96 cross-link remains relatively unaffected. Based on the Kdvalue discussed above, TBP binds strongly enough to cisplatin-modified DNA to protect the platinated site from NER, but konis also an important parameter to consider. When TBP and CFE were added simultaneously to probe DNA in the NER buffer, repair shielding was diminished. Moreover, when TBP was incubated with the DNA probe for a short period of time, its slow binding rate resulted in loss of repair shielding (Figure 2.7C). These results suggest that there is a competition between TBP and repair proteins for the cisplatin-modified site. It has been estimated that there are 30,000-100,000TBP molecules in a mammalian cell, which is similar to the level of HMGB1 (69,70). RPA is also an abundant protein in human cells, being expressed in the range of 30,000-200,000 copies (71). Interestingly, a recent study reported that TBP is 5-10-fold more highly expressed at the protein level in testicular compared to other tissues (72). 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Duplex DNA probes and abbreviations Abbreviation Probes a 5'-AAGGGGGGCTATAAAAGGGGGTGGG- TATAMLP 3' 3'-TTCCCCCCGATATTTTCCCCCACCC-5' 5'-CCTCTCCTCTCAGGATCTTCTCTCC3'-GGAGAGGAGAGTCCTAGAAGAGAGG- 3' 5' 5'-CCTCTCCTCTCAGGTTCTTCTCTCC- 3' AGGT 3'-GGAGAGGAGAGTCCAAGAAGAGAGG- 5' AGGC 5'-CCTCTCCTCTCAGGCTCTTCTCTCC3'- GGAGAGGAGAGTCCGAGAAGAGAGG- 3' 5' 5'-CCTCTCCTCTCTGGATCTTCTCTCC- 3' 3'-GGAGAGGAGAGACCTAGAAGAGAGG- 5' AGGA TGGA 5'-CCTCTCCTCTCTGGTTCTTCTCTCC -3' TGGT TGGC CGGA CGGT CGGC 3 '-GGAGAGGAGAGACCAAGAAGAGAGG- 5' 5'-CCTCTCCTCTCTGGCTCTTCTCTCC- 3' 3'-GGAGAGGAGAGACCGAGAAGAGAGG- 5' 5'-CCTCTCCTCTCCGGATCTTCTCTCC- 3' 3'-GGAGAGGAGAGGCCTAGAAGAGAGG- 5' 5'-CCTCTCCTCTCCGGTTCTTCTCTCC- 3' 3'-GGAGAGGAGAGGCCAAGAAGAGAGG- 5' 5'-CCTCTCCTCTCCGGCTCTTCTCTCC- 3' 3'-GGAGAGGAGAGGCCGAGAAGAGAGG- 5' aBoldface font denotes the TATA box, cisplatin d(GpG) lesion (N1 GGN2) and flanking sequences. 103 Table 2.2. Calculated and observed (ESI-MS)molecular weights for platinated oligonucleotides __ __ Probe Calculated MS (amu) a ESI-MS (amu)b AGGA 7659 7658.66 (0.68) AGGT 7650 7649.79 (1.23) AGGC 7635 7634.13 (1.18) TGGA 7650 7650.46 (1.13) TGGT 7641 7640.70 (0.81) TGGC 7626 7625.88 (0.68) CGGA 7635 7634.02 (0.43) CGGT 7626 7626.22 (1.08) 7611 7610.29 (0.65) CGGC I __ l only for platinated top strands. b one standard deviation is given in parentheses. 104 Table 2.3. Kinetic and thermodynamic parameters for TBP binding to each probe Probe kon (M-ls -1) koff(s -1) Kd (nM) TATAMLP 1.3 + 0.2 x 105 0.57 + 0.05 x 10-4 0.44 AGGA 3.0 + 0.5 x 105 0.89 ± 0.09 x 10-4 0.30 AGGT 2.1 ± 0.2 x 105 0.96 + 0.04 x 10-4 0.46 AGGC 2.3 + 0.1 x 105 1.1+ 0.1x 10-4 0.48 TGGA 1.6 + 0.2 x 105 4.5 + 0.0 x 10-4 2.8 TGGT 1.5 ± 0.1 x 105 3.0 + 0.1 x 10-4 2.0 TGGC n/a n/a 12 ± 1.0 b CGGA 1.3 ± 0.1 x 105 2.6 + 0.2 x 104- 2.0 CGGT 1.2 + 0.2 x 10 5 2.2 + 0.3 x 10 4- 1.9 CGGC n/a n/a 10 a 1.0 b Values indicate average and one standard deviation of at least three experiments. b Measured by competition assay (see text). a 105 A Association 0 40 80120 160 200 240 300 420 600 900 1200 time (sec) ,,... _ " " %no __ TBP-AGGA complex 4- ,_ _~ +_,·FreeAGGA I4-tw NW 0 1 :__ *· . *R h Zc# ft V-0 r0 ftII0sme4WO 00I*too 4- TBP-TATAMLP complex Free to TATAMLP aiD B 0.6 0 0 0.5 0 S 6 0.4 6 o 0 0.3 · 0.2 o 0 x 0 0.1 .0 0 a .. 0 . -..... - - . . . . I . -.. -........................................ -... - 200 400 . - . . . . . . -........... -................................. 600 200 Time (sec) 0 o 0 800 . - . - . - . - 1000 . - . - . - . - 1200 . - . - 1400 Time (sec) Figure 2.1. EMSA experiment to determine kon.(A) TBP-AGGA and TBP-TATAMLP association reactions are represented in the top and bottom autoradiographs, respectively. Free DNA and the TBP complex are shown at various time points and indicated by arrows. (B) The binding fraction (0) for AGGA (closed circles) and TATAMLP (opened circles) are plotted versus time. Lines obtained by fitting of the raw data within 200 sec to eq 1 are shown in the insert. 106 A Dissociation time (min) 0 5 10 20 30 _ -_ 35 50 _ 65 80 m, ,_ 100 120 TBP-AGGA - _ complex oklyl)QIr l- -YN 0 10 20 U~4 -w 30 40 60 lor41Free AGGA 80 100 120 TBP-TATAMLP complex lft..-,A, oMa.. - LFree ,,TAM I- 4 I-I 4- TATAMLP B 0.0 -0.20 o I;11 X a) 'a Q X . -0.40 E E 0. 0 0 C. C -0.60 -0.80 -1.0 0 20 40 60 80 100 120 Time (min) Figure 2.2. EMSA experiment to determine kff. (A) TBP-AGGA and TBP-TATAMLP dissociation reactions are represented in the top and bottom autoradiographs, respectively. (B) The natural logarithm of the complex concentration divided by the initial concentration at t = 0 is plotted versus time. Lines were plotted according to eq 2. 107 ,p C, CPY~to* C 1 L- 0.8 O O&o-C CC *0 6 00 ''"5titMj'-I 1.2 t & C CO 41C atpi Pb W. 1-1-.AAAMA o CD tz 0.6 C 0.4 0) ._ ._ 0.2 0 -1 IF (< CD I- U CD CD C CD ~C < F- U CD CD CD CD I- H H Figure 2.3. EMSA of TATAMLP and cisplatin-damaged < CD CD H( CD CD U C CD probes with TBP. 1-2 nM of ten different probes were allowed to bind to -15 nM of TBP in binding buffer (Experimental procedures). The relative probe binding fractions, normalized to AGGA binding, are represented in bar graph form (bottom). Each value was calculated from three independent experiments and one standard deviation is presented by the error bar. 108 A 5 rx' X a) E 0o 4 0) .0 0 .0 . L. o 3 0. s _ 0L-o r01 X. X 2 0 - Li 0 50 0 B B 150 100 200 Time (sec) U -0.5 vX a) _ a) * -1 EE 00 -1.5 _,) 0 10 20 30 40 50 60 70 Time (min) Figure 2.4. Kinetic EMSA data for cisplatin-damaged probes binding to TBP. (A) Gel profiles for the association reactions were quantified and fit to eq 1 for six cisplatindamaged probes. The fitting data within 200 sec are plotted. (B) Dissociation reactions over 1-hr are fit to eq 2 for each probe. Probes are: AGGT (closed circles), AGGC (open circles), TGGA (closed triangles), TGGT (open triangles), CGGA (closed squares), and CGGT (open squares). Results for 3 or 4 trials were averaged and one standard deviation is indicated by the error bars. 109 A I TGGC (nM) 600 0 im_ |0 tW I dla~/~l-i B TBP-TATAMLP _ complex /-- l -TATAMLP Free 1.0 0.75 c- 0 L_ in 0.50 C ,0.25 nfl 0.1 1 10 100 1000 10000 [TGGC] (nM) Figure 2.5. Competition EMSA assay between TGGC and TATAMLP. (A) A representative competition assay in which 1.4 nM of TATAMLP and 2 nM of TBP were mixed with increasing amounts of TGGC. (B) Plot of the binding fraction against the concentration of TGGC (0-600 nM). C1/2, the TGGC concentration at 0=1/2, is indicated by a dotted arrow. 110 2 nM TBP 1 nM TBP ,,,.------- f AGGA (nM) 20 0 12 0 TBP-TATAMLP complex 4- 123456789 1 2 -ii 1e 3 4 5 6 7 8 10 20 10 11 12 13 14 9 Free TATAMLP I.- C J V 4. 1o 1 0.8 rC' 4. 'C C 5 0.6 5 0C rv C 4. C: 0.4 a . 5 0.2 0 0 1 2 4 8 - 0 1 2 4 8 12 [AGGA] (nM) Figure 2.6. Competition EMSA assay between AGGA and TATAMLP. Lane 1 contains the TATAMLP probe without TBP. A 4 nM solution of TATAMLP and 2 nM (lane 2-8) or 1 nM (lane 9-14) of TBP were mixed with increasing amounts of AGGA as indicated along the abscissa in the bar graph at the bottom. The relative binding fractions of lanes 2-14 are shown in this graph. At each TBP concentration, all lanes are normalized to the binding fraction in the absence of AGGA (lanes 2 and 9). 111 Figure 2.7A GG [TBP] (pM) 0 20 50 GTG 100 0 20 50 100 30 nt- 24 nt- -1111 1W *9.RP . .wlffi-e 88, ;.r; -'s,? -q a: ·.i: StyW Wr* 112 Figure 2.7B 0 .h Cu CL M, bQI V OL TBP concentration (pM) Figure 2.7C TBP-shielding time course 100.00 r 80.00- I- . 60.00 ) 40.0020.00- N an u.Uu 0 10 20 30 40 50 60 70 TBP incubation time Figure 2.7. Effect of TBP on excision repair assay of cisplatin-DNA intrastrand crosslinks in HeLa cell extract. The substrates were pre-incubated for 30 min prior to addition of HeLa CFEs. (A) 10% denaturing gel demonstrating the diminishing repair signal by TBP. (B) Densitometric quantification of the data. Relative repair levels are plotted against the concentration of TBP added using 1,2-d(GpG) (circles) and 1,3d(GpTpG) (triangles) intrastrand cross-links as repair probes. (C) Incubation timedependence of repair shielding by TBP. The substrates were pre-incubated for indicated time prior to addition of HeLa CFEs. Error bars represent one standard deviation. The absolute excision levels in the absence of TBP were 0.2 0.05% for the 1,2-d(GpG) intrastrand cross-link and 2.0 + 0.5%for the 1,3-d(GpTpG) intrastand cross-link. 113 Figure 2.8A 3' G G rp rl -+ + (-Ic G G G A A A A T A *-w *ow-; T -~ C G G G G G .x v A A urn I 3'4-- 5' 114 Figure 2.8B 3' G A G A - -+ G U A G A G T C C T U_. .- ., -O \ .. -N+.e G A G A A G A G G 3' *- 5' Figure 2.8. Hydroxyl radical footprint of TBP. (A) TBP bound to the top strand of the TATAMLP. (B) TBP on the non-damaged strand of the AGGA duplex. The dotted line is the profile for a control sample (-) in the absence of TBP. The solid line indicates the profile for the footprint with TBP (+). The protected region is indicated in boldface font. 115 A , 5 '-AAGGGGGGC T ATAAAA#GGGGGTGGG- 3' 3'-TTCCCCCCGAtTATTTT CCCCCACCC- 5' B , 5'-GGAGAGAAGAXCCX GAGAGGAGAGG-3' 3'-CCTCTCTTCTN 2 GGN1 CTCTCCTCTCC-5' Figure 2.9. Schematic diagram of TBP-DNA complex interactions as revealed by footprinting. (A) TBP-TATAMLP interaction. Boldface font indicates the conserved TATA box. Intercalating phenylalanine residues and hydrogen-bonding contact regions (19) are indicated by arrows and closed circles, respectively. (B) TBP-cisplatin-damaged DNA interaction. Boldface indicates the cisplatin d(GpG) lesion and flanking sequences. Dotted and solid braces show regions protected by TBP from DNase I (48) and hydroxyl :radical (this study), respectively. 116 Chapter 3 The Nature of Full-Length HMGB1 Binding to Cisplatin-Modified DNA * The work in this chapter has been published in Biochemistry,2003,42, 2664-2671. 117 Introduction HMGB1 is an abundant and highly conserved high mobility group (HMG) chromosomal protein. HMGB1 binds preferentially without sequence specificity to bent or distorted DNA structures such as those at four-way junctions (4WJs) and cisplatin damage and subsequently bends the target DNA (1). This non-histone DNA binding protein appears to act mainly as an architectural facilitator in the assembly of nucleoprotein complexes. Although the exact roles of HMGB1 are not fully defined at present, HMGB1 is involved in DNA transcription and recombination (1). In addition, :recent studies have revealed critical extracellular roles for HMGB1. The protein is secreted by certain cells where it mediates such processes as inflammation, differentiation, migration and tumor metastasis (2,3). HMGB1 consists of two tandem HMG-box domains (A, B) and a C-terminal acidic tail (Figure 3.1). Both A and B domains of HMGB1 share a common HMG box structure. Three a-helices arranged in the shape of an L comprise the 80-amino acid domain motif, which largely determines the DNA-binding properties of HMGB1 (4). The boxes are very similar but not identical. Domain A has a higher binding affinity to distorted DNA than domain B, whereas domain B can bend DNA more effectively. Because of the importance of the HMG box interaction with DNA, many researchers have focused on the structures of the individual HMG domains and their DNA complexes. These studies have clarified the structural basis of DNA binding and bending of the HMG box (5). Significantly less information is available about full-length HMGB1 and its DNA binding properties, however. The reason why HMGB1 contains two HMG boxes having almost identical properties is currently unclear. In one study, it was demonstrated that both HMG boxes of HMGB1 are required for enhanceosome formation with the ZEBRA protein (6). 118 Generally, each HMG box and the basic linker regions between them contribute to the DNA-binding properties of the AB didomain (7-9). An intriguing feature of HMGB1 is its unusually acidic C-terminal tail, which is highly negative and contains a run of 30 consecutive aspartic and glutamic acids. The net charge of HMGB1 (pI = 5.0) is thus quite different from that of the AB didomain (pI = 10), solely because of the acidic domain. This C-terminal tail modulates the DNA binding of HMGB1, reducing binding affinity in most cases (10). Although the acidic tail provides HMGB1 with properties distinct from the HMG boxes, little is known about the structural properties and functional basis of this domain. Cisplatin is one of the most widely used anti-cancer drugs. It manifests its cytotoxicity to tumor cells by damaging DNA through the formation of covalent bonds to the purine bases (11). Both domains A and B as well as the full-length HMGB1 protein selectively bind with high affinity to the major d(GpG) and d(ApG) 1,2intrastrand cross-links formed by cisplatin on DNA. Several reports suggest that the interaction between HMGB1 and cisplatin-damaged DNA can contribute to its biological activity (12-14).The recent discovery of multiple roles for HMGB1 both in the nucleus and as an extracellular signaling protein further support the possible involvement of HMGB1 in mediating cisplatin action in cancer cells. The interaction of individual domains A and B with cisplatin-modified DNA has been extensively studied by a variety of techniques including X-ray crystallography (15,16).The complex formed by the full-length protein with cisplatin-modified DNA has not been extensively examined, however. The effect of the presence of tandem HMG boxes and the acidic Cterminal region of HMGB1 on the interaction with cisplatin-modified DNA has, until the present study, been unknown. 119 Here we present various experiments, including electrophoretic mobility shift assays (EMSAs), footprinting, and EDC cross-linking, designed to examine the binding mode of HMGB1 to DNA modified site-specifically by cisplatin. The results provide the first structural information of the interaction between full-length HMGB1 and cisplatinmodified DNA. In addition, the interaction of the C-terminal tail with the rest of the protein and its effect on HMGB1 binding to cisplatin-modified DNA were investigated. These studies provide insight into the structure-specific DNA binding properties of HMGB1. Experimental Procedures Construction of Expression Vectors.The peptide fragments corresponding to fulllength HMGB1, domain A, domain B, and didomain AB165 are depicted in Figure 3.1. The cDNAs of HMGB1 (residues 1-215), didomain AB165 (residues 1-165), domain A (residues 1-89), and domain B (residues 86-165) were amplified by PCR from the plasmid pT7-RNHMG1 (provided by M. E. Bianchi) containing the rat HMGB1 cDNA as a template. All amplified cDNAs were cloned into the expression vector pET32Xa/LIC (Novagen) except for the HMGB1 cDNA, which was cloned into pET30Xa/LIC (Novagen). Both expression vectors contain encode a His-tag fusion peptide at the N-terminus of the target protein. Site-directed mutagenesis was performed to mutate intercalating residues of HMGB1 and AB165 according to the QuikChange protocol (Stratagene). Expression and Purification of HMGB1 Proteins. All HMGB1 proteins were expressed in BL21(DE3)cells. Cell cultures were shaken at 200 rpm and 37 °Cuntil the optical density, OD600, was 0.5 - 0.7. Isopropyl-13-D-thiogalacto-pyranoside (IPTG) was then added to the cell culture at a final concentration of 50 pM and cells were grown for 120 another 16 hr with shaking at 150 rpm and room temperature (23 - 30 °C). All His-tag fusion proteins were purified by a His-bind (Novagen) column according to the manufacture procedure. The fusion peptides containing a His-tag were cleaved by Factor Xa treatment, yielding intact target proteins. A Macro-prep High S column (BioRad) was used to purify further all proteins except for full-length HMGB1, which was purified on an anion-exchange column (Sepharose CL-6B, Pharmacia Biotech) because of its highly negative C-terminal tail. Pure proteins were dialyzed against storage buffer (10 mM Tris pH 7.5, 100 mM NaCl, 1 mM EDTA, 2 mM -mercaptoethanol, 0.2 mM PMSF) and stored at -80 °C before use. Preparationof OligonucleotidesProbes.Figure 3.1Bshows the oligonucleotides used in this work together with their abbreviations. The oligonucleotides were synthesized, platinated, and purified as previously described (17).The purity and composition of the probes containing cisplatin adducts were confirmed by HPLC, UV/Vis spectroscopy and electrospray mass spectrometry (data not shown). ElectrophoreticMobility Shift Assay (EMSA). The single-stranded oligonucleotides (-20 pmol) were radioactively labeled at their 5'-ends by using 50 Ci of [- 32P]ATP (PerkinElmer Life Sciences) and 20 U of polynucleotide kinase (New England Biolabs). The labeled oligonucleotides were annealed with complementary strands in 10 mM Tris (pH 7.0), 50 mM NaCl, and 10 mM MgCl2, heated to 90 °C, and slowly cooled to 4 °C over several hours. The labeled duplex probe was mixed with the indicated amount of protein in binding buffer (10 mM HEPES pH 7.5, 10 mM MgC12, 50 mM LiCl, 100 mM NaC1, 1 mM spermidine, 0.2 mg/ml BSA, 0.05% Nonidet P40). The binding mixtures were incubated on ice for 30 min before loading onto 10% native polyacrylamide gels. 121 Gels were electrophoresed at 300 V for 1.5 hr at 4 °C in 0.5 x TBE, followed by geldrying and autoradiography. Footprinting Assay. The platinated DNA duplex, 35AGGA (-50,000 cpm), in which only a single-strand was labeled at the 5'-end phosphate, was mixed with the indicated amount of proteins in the same buffer used in EMSA. The total binding solutions, 15 pl, were incubated on the ice for 30 min, then subjected to the hydroxyl radical reaction. Cleavage reactions were initiated by adding 2 ll of 100 mM ascorbate, 2 lI of 1.5% H2 0 2, and 2 1Iof 50 mM Fe(II) EDTA to the binding solutions. After the 4 min reaction period at room temperature, 10 1Iof 1 M thiourea was added to quench the reactions. Cleaved oligonucleotides were isolated by ethanol precipitation and analyzed on a 20 % polyacrylamide denaturing gel. EDC Cross-Linking. The indicated dimethylaminopropyl)carbodiimide) amount of EDC (1-ethyl-3(3- was allowed to react with HMGB1 (5 tM) in the presence or absence of 25TGGA (7 tM) for various times at room temperature in reaction buffer containing 50 mM MES pH 5.3 and 20 mM NaCl. The reactions were quenched by adding 13-mercaptoethanolto a final concentration of 20 mM. Cross-linked proteins were directly analyzed by SDS-PAGE or stored at -20 °C for further study. Cyanogen bromide (CNBr) cleavage reactions were performed in 70% formic acid containing 250 mM CNBr by adding the solution to a lyophilized HMGB1 sample. Produced peptide fragments were separated by a 16.5% Tris-tricine gel (Bio-Rad), transferred to a PVDF membrane, and stained by amido black 10B. N-terminal sequencing was carried out for each peptide fragment band on the membrane by the MIT Biopolymers Laboratory. 122 Results Footprinting Analysis of HMG Box Protein Binding to Cisplatin-Modified DNA. Footprinting is a very powerful methodology for defining the binding mode of a DNAprotein complex. The hydroxyl radical footprinting assay was used to examine the interaction of four HMGB1 proteins, dom A, dom B, AB didomain, and full-length HMGB1 with cisplatin-modified DNA (Figure 3.2). In this paper, dom A and dom B denote individual domains A (residues 1-89) and B (residues 86-165), as shown in Figure 3.1A. Protection regions in the vicinity of the platinated 1,2-d(GpG) cis{Pt(NH3) 2}2 + cross-link by dom A and dom B are consistent with previous results (18). A 5-base pair (bp) fragment extending to the 3' side of the 1,2-d(GpG) cross-link site reveals the footprint of dom A, whereas dom B affords symmetric protection with respect to the platination site (Figure 3.2). Of significance is that DNA fragments protected from hydroxyl radicals by HMGB1 and AB165 are almost identical to that of dom A, with no detectable domain B protection pattern around the damage site. Mutation of IntercalatingResidues in HMGB1. The binding properties of HMGB1 proteins to cisplatin-modified DNA were examined by EMSA. Figure 3.3 shows binding titrations of HMGB1 and the AB didomain to the site-specifically platinated duplex DNA (25TGGA). Probes of different lengths (15, 20, 25, and 35 bp) were investigated. Under our experimental conditions, >25 bp probes showed efficient binding for fulllength HMGB1 (data not shown). Apparent dissociation constants, Kd, were calculated from the protein concentrations when half of the DNA probes form protein-DNA complexes (Table 3.1). Previous work revealed that intercalating residues are critical for tight binding of HMG domains of HMGB1 to cisplatin-modified DNA (Figure 3.1B) (18). Mutation of Phe37 to Ala in dom A dramatically abolishes its DNA binding affinity. The dom B 123 double mutation, F16A and I37A, also reduces its binding affinity, by more than 25-fold. In order to explore further the roles of individual domains of HMGB1 in the interaction with cisplatin-modified DNA, the intercalating residues of each domain in the HMGB1 full-length protein and on the didomain were mutated. HMGB1 F37A, in which Phe37 of domain A is converted to Ala, exhibits weakened binding affinity toward 25TGGA compared to wild type HMGB1. On the other hand, HMGB1 F102A I121A, a double mutant of the intercalating residues of domain B, has almost same the dissociation constant as that of HMGB1 (Figure 3.4A, Table 3.1). Moreover, as revealed by Figure 3.4B, AB165 F37A has the weak binding affinity compared to wild type AB165 and AB165 F102A I121A. These data reinforce the conclusion of the footprinting results, :namely, that domain A of both HMGB1 and the AB didomain solely mediates the interaction with cisplatin-modified DNA. The hydroxyl radical footprinting assay was also performed to investigate how mutation of an intercalating residue in each HMG domain might alter the binding mode of HMGB1 and its didomain to cisplatin-modified DNA. As shown in Figure 3.5, F37A mutants of both proteins protect the DNA fragment symmetrically about the cisplatin lesion, like dom B (Figure 3.2). These results are consistent with symmetric binding by site domain B of these mutated proteins to the cisplatin damage site. Although we cannot formally exclude the possibility that mutated dom A now binds symmetrically in the mutant protein complexes, the fact that the F37A of dom A shows almost no interaction with cisplatin-modified DNA (16,18) strongly supports our interpretation. Protection by the F102A I121A double mutant does not differ from that of wild type protein (data not shown), as expected. Effectof the C-terminalAcidic Tail. The footprinting and mutation studies indicate that both HMGB1 and didomain AB interact with cisplatin-modified DNA through 124 their A domain. On the other hand, our results also reveal the binding properties of fulllength HMGB1 to be distinct from the didomain, which lacks the acidic C-terminal tail, as reported in previous literature (10).The interaction of the C-terminal tail with the rest of the HMGB1 protein was therefore analyzed by a cross-linking experiment. EDC, a zero-length cross-linking agent, can react with carboxyl and amino groups of protein side chains. As displayed in Figure 3.6A, HMGB1 is cross-linked by EDC to an extent that increases with the concentration of the carbodiimide. Cross-linked HMGB1 migrates faster than intact HMGB1 in SDS-PAGE. The cross-linking requires the presence of the C-terminal tail since the didomain AB shows no such cross-linked bands in the gel (data not shown). Cross-linked HMGB1 was further characterized by CNBr cleavage, a reaction that cuts peptide bonds after methionine (Met) residues (19). Following CNBr cleavage reactions of intact and cross-linked HMGB1 proteins, the produced peptide fragments were separated by SDS-PAGE and assigned by amino acid sequencing (Figure 3.6B). HMGB1 contains 6 Met residues including the N-terminus (Figure 3.6C). The bands corresponding to peptide fragments cross-linked by EDC, which are distinguished by showing cleavage only among cross-linked HMGB1 samples (Figure 3.6B; lanes 2-4), are indicated by brackets in the gel. In the case of the sample containing partially cross-linked HMGB1 (Figures 3.6A and 3.6B; lane 2), only fragments 133-215, which contain a C-terminal tail, and fragment 76-132 are crosslinked with each other by EDC. The complex of HMGB1 with cisplatin-modified DNA was similarly treated by EDC and compared with EDC-treated HMGB1 alone. Upon binding to platinated DNA, HMGB1 displayed a higher degree of cross-linking than did free HMGB1 (Figure 3.7A); the CNBr cleavage patterns were almost identical, however (data not shown). Moreover, as shown in Figure 3.7B, all such cross-linked HMGB1 proteins remain 125 complexed with cisplatin-modified DNA, like intact HMGB1. EDC-treated HMGB1 binding to cisplatin-modified DNA was also examined (Figure 3.8). HMGB1 crosslinked with a low concentration of EDC (HMGB1_EDCI) interacts with cisplatinmodified DNA similar to intact HMGB1 (HMGB1_NoEDC). Cross-linking with a high concentration of EDC, however, abolishes HMGB1 binding to cisplatin-modified DNA. Discussion Full-LengthHMGB1 Binding to Cisplatin-ModifiedDNA; Two Tandem HMG Boxes. More than 120 proteins in the cell contain an HMG box motif (20). Many such proteins :from a variety of organisms contain a single HMG box domain (HMG-D, NHP6A/B, SRY,SOX, and LEF1), although some have up to six copies of HMG boxes, such as UBF (upstream binding factor). The mechanism by which proteins containing multiple HMG boxes bind to target DNA and the roles of these many domains remain unresolved. The hydroxyl radical footprinting and mutagenesis results presented here reveal that HMG(;B1and its didomain protein bind to cisplatin-modified DNA, primarily but not exclusively through domain A. As shown in Figure 3.2, HMGB1 and the AB didomain protect DNA around a cisplatin-damaged site in an asymmetric fashion. The protected region is identical to that afforded by domain A alone. This asymmetry argues strongly for a structure closely resembling that of domain A bound to a DNA 16-mer containing the cis-{Pt(NH3) }2 2+ intrastrand d(GpG) cross-link (16). Moreover, there is no detectable DNA protection by domain B of HMGB1 or the didomain, which gives a symmetric footprint (18). With domain A covering the cisplatin site in these proteins, domain B has a chance to interact only with residual DNA surrounding the cisplatin-damaged site, which structurally resembles normal DNA (16). Such an interaction might be too weak to protect DNA from the hydroxyl radical reaction. We cannot rigorously exclude the 126 possibility of domain B interactions with DNA around the cisplatin site, however. In a recent example, domain A of the AB didomain also dominated the interaction with 4WJ DNA, while domain B bound to one of the arms of the junction (21). Our site-directed mutagenesis experiments provide additional evidence that domain A mediates HMGB1 binding to cisplatin-modified DNA. When the key intercalating residue Phe37 of domain A in HMGB1 is converted to Ala, the DNAbinding affinity is reduced, whereas the domain B mutation does not affect the binding affinity. In addition, the footprinting assay confirms that the reduced binding affinity of HMGB1 F37A or AB 165 F37A toward cisplatin-modified DNA arises from a new binding mode of these proteins, in which one HMG-box domain protects the cisplatin damage site symmetrically. The footprint patterns of HMGB1 F37A and AB 165 F37A are identical to that of dom B, as shown in Figures 3.2 and 3.5. Moreover, in the dom A F37A mutant, in which Phe37 is replaced by Ala to eliminate the intercalating residue, there is almost no specific interaction with cisplatin-modified over undamaged DNA, as described in previous reports (15,18). Therefore, it is difficult to imagine that the mutated domain A of HMGB1 F37A or AB 165 F37A interacts at the site of platination to produces the clear footprint exhibited in Figure 3.5. The results strongly imply that domain B binds to the cisplatin intrastrand d(GpG) cross-link site in the mutated proteins, HMGB1 F37A and AB 165 F37A. Nonetheless, it is possible that HMG-box domains in full-length HMGB1 or the didomain AB 165 behavior differently from the individual domains. We therefore cannot completely exclude that domain A of F37A mutated proteins interacts with the damage site and displays a symmetric footprint. The dissociation constant of HMGB1 F37A is 210 nM, which indicates diminished binding compared to wild type HMGB1 (Kd= 120 nM). This F37A mutation, however, reduces the binding affinity only by a factor of two. Considering the fact that 127 domain B in HMGB1 F37A is likely bound to cisplatin-damage site, this result is unexpected since previous results revealed dom B (K = 39 nM) to be a much weaker DNA binding protein than dom A (Kd= 1.5 nM) toward cisplatin-modified DNA (15). This behavior is the first manifestation that the HMG box domains in full-length HMGB1 react differently than the individual domains. In the case of the HMGB1 didomain binding to 4WJ DNA, domain B interacts adjacent to a structure-specific binding region whereas domain A binds to the central hole (21). Our results indicate, however, that in mutant (F37A) or wild type full-length .HMGB1,only one HMG box domain interacts strongly with the cisplatin damage site ,(Figures 3.2 and 3.5). In order to investigate the possible interaction of domain B with DNA at the cisplatin site, we synthesized asymmetrically platinated DNA probes, in which the GG cisplatin-damage site was positioned either at 3' side or the 5' side, rather than in the middle of the DNA probe. The interactions of HMGB1 and AB165,however, were unaffected by the positioning of the cisplatin moiety (data not shown). This result indicates either that domain B of HMGB1 is not specifically binding toward one side of the cisplatin locus (3' or 5' side), or that is not interacting at all with the rest of DNA after protein binding through domain A. F37A mutants, where domain B is bound to the cisplatin site, were similarly tested. Again, all three probes showed the same binding affinities toward HMGB1 F37A and AB165 F37A (data not shown). In all cases, only one HMG box of HMGB1 affected the interaction with cisplatin-modified DNA. The C-terminal Tail in HMGB1. Several researchers have tried to delineate the roles of the acidic C-terminal tail of HMGB1 by comparing the properties of the fulllength protein with those of the didomain lacking the C-terminus (8-10). These studies demonstrated that the acidic tail generally reduces DNA binding affinity, as expected from electrostatic consideration. The length of the acidic tail controls the DNA binding 128 affinity of the HMG box proteins HMGB1/ 2. Consistent with these results, we find the AB didomain to display much higher binding affinity, not only for cisplatin-modified DNA, but also for undamaged DNA, compared to full-length HMGB1 (Table 3.1). At present, there is no structural information that reveals how the C-terminal tail modulates DNA binding ability for any HMG box protein. Domain-domain interactions between the acidic C-terminus and the A and B boxes of HMGB1 were therefore studied by cross-linking experiments and CNBr digestion to gain insight into this question. As shown in Figure 3.6, there is no single specific cross-linking site of the C-terminal tail with amino acids of the N-terminal site in HMGB1. The cross-linked HMGB1 proteins appear as a broad band that is inconsistent with a single cross-link site (Figure 3.6A). This result is not unexpected, however, since all 30 amino acids of the acidic tail are capable of forming covalent bonds upon EDC activation. We performed CNBr digestion and amino-acid sequencing to provide a coarse map of the regions containing crosslinking sites of the C-terminal tail in HMGB1. Cyanogen bromide treatment generates five major peptide fragments from unmodified HMGB1 (Figure 3.6C). The cleavage pattern of cross-linked HMGB1 samples (Figure 3.6B) suggests that the C-terminal tail might interact mainly with domain B and the linker regions, and not with domain A. This conclusion is based on the lack of a CNBr-induced peptide fragment corresponding to domain A (14-52)in cross-linked peptide fragments of partially crosslinked HMGB1 (lane 2 in Figure 3.6B).The acidic tail will cross-link with the domain A region of HMGB1 at higher concentrations of EDC, however (lane 4 in Figure 3.6B). These results are consistent with previous differential scanning calorimetry (DSC) experiments of HMGB1, in which the authors proposed that one of the HMG domains in HMGB1 is interacting with the acidic tail (22). According to our data, domain B is most likely the main region for such an interaction with the C-terminal tail. 129 EDC cross-linking studies of HMGB1 in complex with DNA suggest another interesting feature of the C-terminal tail interaction (Figure 3.7). Despite the likelihood that HMGB1 interactions with the target will place protein residues in contact with the DNA, there is even more cross-linking between the tail and the rest of HMGB1 in the complex (Figure 3.7A). The EDC-dependent cross-linking also does not disrupt proteinDNA complex formation (Figure 3.7B). The same experiment was performed with HMGB1 F37A, in which domain B likely interacts with the DNA (data not shown). EDC cross-linking of the HMGB1 F37A-DNA complex gave results almost identical to those of the HMGB1-DNA complex. Taken together, the data suggest that, although domain B and the linker regions are the main interaction sites for the C-terminal tail in HMGB1, the interactions are random in these regions. We also examined the interaction between cisplatin-modified DNA and previously cross-linked HMGB1 (Figure 3.8). Lightly cross-linked HMGB1, in which the C-terminal tail mainly cross-links with domain B and the linker regions, has the same level of binding to cisplatin-modified DNA as intact HMGB1. More highly cross-linked HMGB1, in which the C-terminal tail crosslinks with all of HMGB1 including domain A, however, loses its binding affinity to cisplatin-modified DNA. Implicationsfor the Mechanism of HMGB1 Function. As a non-sequence specific DNA binding protein, HMGB1 alters chromatin structure, represses or activates transcription, and promotes recombination (1). The manner by which HMGB1 mediates these DNA-dependent processes is not well understood. In addition to DNA binding and bending, HMGB1 must recognize some signal by which it locates the target DNA. Our results indicate one manner by which HMGB1 can perform this task. Only one HMG box is essential for binding to the specific DNA structure produced by cisplatin 1,2-intrastrand d(GpG) cross-link. The rest of HMGB1, domain B and the C-terminal 130 tail, might serve to guide HMGB1 to the target through interaction with other sequencespecific DNA binding proteins. HMGB1 increases the binding affinity of various DNA binding proteins including steroid hormone receptors, TBP, and RAG1/2 (23-25). In addition, HMGB1 appears to interact directly with these proteins in vitro. A good example, in which HMGB1 assists V(D)J recombination, was recently reported (26). In this study, HMGB1 stimulated RAG protein binding to a target signal, recombination signal sequence (23-RSS),by direct interaction with RAG. At the same time, HMGB1 helped catalyze cleavage of 23-RRSthrough the direct binding. HMGB1 is thus able to interact with DNA and other proteins simultaneously. The enhancement of ZEBRAbinding to its promoter site by HMGB1 requires two tandem HMG boxes. In contrast, only one HMG box of HMGB1 is sufficient to stimulate Rta dimer binding to a target gene (27). The mechanism by which HMGB1 decides to use only one or two of its HMG boxes to perform such roles remains to be elucidated. As discussed above, HMGB1 F37A, binding through domain B to cisplatin-modified DNA, shows only slightly weak interaction compared to wild type HMGB1, which binds through domain A. In full-length HMGB1, domain B is capable of interacting in a structure-specific manner, whereas domain A has a slightly higher binding affinity. Small DNA minicircles provide the best example (28). Unlike cisplatin-modified DNA, DNA minicircles have a non-localized distorted structure that extends over the entire duplex. Therefore all regions of the minicircle can serve as a binding locus for the HMG box domain. HMGB1 exhibits much higher binding affinity for DNA minicircles (Kd = -2 nM) than cisplatin-modified DNA (Kd = 120 nM), whereas domain A alone has similar binding affinities to both kinds of DNA. The tandem HMG boxes of HMGB1 are probably each bound to the DNA minicircles. In the cooperative binding of ZEBRAand HMGB1 to the target promoter, the complex generates 14-bp bent DNA at the HMGB1 131 binding site (6). This long piece of distorted DNA is a suitable binding site for both HMG boxes of HMGB1. Two tandem HMG boxes and the unusual C-terminal tail make it possible for HMGB1 to perform more roles than would be possible for a single domain. Depending on the function, the target DNA, and the requirement of other proteins, HMGB1 is able to use its multiple domains. Roles of HMGB1 in Cisplatin Action. Cisplatin manifests its cytotoxicity by damaging DNA, generating a distorted DNA duplex. A number of cellular proteins can interact with this structurally altered DNA and affect the processing of cisplatin-DNA lesions in the cell (29,30). Among these proteins, HMGB1 has for many years been a subject of much study, but a strong link of this protein to the cisplatin mechanism of action is lacking. Many reports demonstrated that different levels of HMGB1 can affect the processing of cisplatin-DNA lesions in the cell (12,14,31-33).In the present study, HM(GB1displayed a high binding affinity toward cisplatin-damaged DNA (Kd = 120 nM) under physiological conditions. Its high abundance in the cell and high affinity support an involvement in cisplatin action. On the other hand, the fact that only one HIMGbox interacts strongly at the cisplatin site suggests the possibility that the remainder of protein will interact with other cellular components. Since HMGB1 interacts with many such proteins in the cell (34), it possibly recruits other factors that can affect cisplatin processing in certain cell lines. The exact roles of HMGB1 in mediating cisplatin cytotoxicity must therefore be investigated by considering its multiple roles and different expression levels in a variety of cell types. 132 Conclusion This work provides structural insight into the interaction of HMGB1 with cisplatin-modified DNA. Only one of the two tandem HMG boxes controls the interaction. Full-length HMGB1 protein and its didomain lacking the acidic C-terminus bind to DNA containing a site-specific cisplatin 1,2-intrastrand d(GpG) cross-link, mainly through domain A. Mutation of the key intercalating residue Phe37 of domain A to Ala in full-length HMGB1 alters the binding mode of the protein, possibly allowing domain B to bind to the site of cisplatin damage. This mutant protein (HMGB1Phe37A), however, has only slightly diminished binding affinity for cisplatin-damaged DNA compared to wild type HMGB1, whereas domain B alone binds much more weakly than domain A alone. This result suggests that HMG-box domains in full-length HMGB1 might have different DNA binding properties from those of the isolated individual HMG-box domains. EDC cross-linking experiments reveal that the acidic Cterminal tail cross-links mainly with domain B and the linker regions in HMGB1. Interactions of the C-terminus with these regions are non-specific, however, and the Cterminus can also cross-link with domain A at high levels of EDC. Our results thus emphasize the importance of studying full-length HMGB1 containing two tandem HMG boxes and the acidic tail rather than a single HMG box. 133 References 1. Thomas, J. O., and Travers, A. A. (2001) Trends Biochem. Sci. 26, 167-174 2. 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(1996) Biochemistry 35, 10004-10013 14. Brown, S. J., Kellett, P. J., and Lippard, 15. Dunham, 16. Ohndorf, U. M., Rould, M. A., He, Q., Pabo, C. O., and Lippard, S. J. (1999) S. U., and Lippard, S. J. (1993) Science 261, 603-605 S. J. (1997) Biochemistry 36, 11428-11436 Nature 399, 708-712 17. Jung, Y., Mikata, Y., and Lippard, 18. He, Q., Ohndorf, S. J. (2001) J. Biol. Chem. 276, 43589-43596 U. M., and Lippard, S. J. (2000) Biochemistry 39, 14426-14435 134 19. Kaiser, R., and Metzka, L. (1999) Anal. Biochem. 266, 1-8 20. Baxevanis, A. D., and Landsman, D. (1995) Nucleic Acids Res. 23, 1604-1613 21. Webb, M., and Thomas, J. 0. (1999) J. Mol. Biol. 294, 373-387 22. Ramstein, J., Locker, D., Bianchi, M. E., and Leng, M. (1999) Eur. J. Biochem. 260, 692-700 23. Boonyaratanakornkit, V., Melvin, V., Prendergast, P., Altmann, M., Ronfani, L., Bianchi, M. E., Taraseviciene, L., Nordeen, S. K., Allegretto, E. A., and Edwards, D. P. (1998) Mol. Cell. Biol. 18, 4471-4487 24. Das, D., and Scovell, W. M. (2001) J. Biol. Chem. 276, 32597-32605 25. Fugmann, S. D., Lee, A. I., Shockett, P. E., Villey, I. J., and Schatz, D. G. (2000) Annu. Rev. Immunol. 18, 495-527 26. Swanson, 27. Mitsouras, K., Wong, B., Arayata, C., Johnson, R. C., and Carey, M. (2002) Mol. P. C. (2002) Mol. Cell. Biol. 22, 1340-1351 Cell. Biol. 22, 4390-4401 28. Webb, M., Payet, D., Lee, K. B., Travers, A. A., and Thomas, J. 0. (2001) J. Mol. Biol. 309, 79-88 29. Kartalou, M., and Essigmann, 30. Zlatanova, J., Yaneva, J., and Leuba, S. H. (1998) FASEB J. 12, 791-799 31. Huang, J. C., Zamble, D. B., Reardon, J. T., Lippard, J. M. (2001) Mutat. Res. 478, 1-21 S. J., and Sancar, A. (1994) Proc. Nat. Acad. Sci. USA 91, 10394-10398 32. Nagatani, G., Nomoto, M., Takano, H., Ise, T., Kato, K., Imamura, T., Izumi, H., Makishima, 33. K., and Kohno, K. (2001) Cancer Res. 61, 1592-1597 Wong, B., Masse, J. E., Yen, Y. M., Giannikopoulos, P., Feigon, J. J., and Johnson, R. C. (2002) Biochemistry 41, 10182-10192 34. Dintilhac, A., and Bernues, J. (2002) J. Biol. Chem. 277, 7021-7028 135 Table 3.1. Affinities of HMGB1 Proteins Toward Cisplatin-Modified Probe Kd(nM) HMGB1 25TGGA 120 + 10 HMGB1 F37A 25TGGA 210 + 15 HMGB1 F102A I121A 25TGGA 115 + 5 25TGGANoPtb > 1000 Protein HMGB1 AB165 25TGGA 0.5 + 0.2 AB165 F37A 25TGGA 1.2 + 0.3 25TGGA 0.4 + 0.2 -- AB165 F102A I121A AB165 25TGGANoPtb DNA a 30 + 20 aValues and one standard deviation of at least three experi ments. ---- average -. indicate Co--- .t b'25TGGA_NoPtindicates the same probe as 25TGGA without cisplatin damage. 136 A B 1 HMGB1 215 C N ~ 1 AB165 N dom A N 11 ~ L 1 25TGGA 5' -CCT'CTCC'CTCTGGATCT'TTCTC'CC-3 ' 3' -GGAGAGGAGAGACC AGAAGAGAGG-5' 165 C N C 89 1 C 86 dom B N 35AGGA 165 C 5'-TCCTCTCTCTCCTCTCAGGATCTTCTCTCTCTTCC-3' 3'-AGGAGAGAGAGGAGAGTCCTAGAAGAGAGAGAAGG-5' C 10 1 HMGBP6 domA NHP6A HMG-D LEF1 SRY 20 30 GKGDPKKPRGK.MS SiFFVQTCREEHKH 40 PDASVNSEFSXKC 50 60 80 70 SERWKTMSAEKGKEDMAKADKARYREMKTY I PPKGETKXK MVTPRE PKKRTTRKKKDPNAPKRALSAY FFANENRD IVRSENPD I T- -EGQVGKKLGEKWKALTPEEKQ PYEAKAQADKKRYESEKELYNAT LA MSDKPKRPLSAl LWLNSARES IKRENPGI K - TEVAKRGGELWRAM- - KDKSEWEAKAAKAKDDYDRAVKEFEEANGGS SAANGGGAKKR MHI KKPLNAFLYMKEMRANVVAECTLKE -AAINQI LGRRWHALSREEQAKYYELARKERQLHMQLY PGWSARDNYGKKKKRKREK QDRVKRPMNATIVtS RDQR- RKMAENPRMR SE I SQLGYQWMLTEAEKWPFFQEAQKLQAMHREKYPNYK HMGB1 domB Acidic tail FKDPNAPKRP PSA:FCSEYRPK 90 100_ 110 IKGEHPGLS - -FI DVAXKLGEMWNNTAADDKQPYEKKAAKLKEKYEKDIAAYRAK 120 130 140 150 160 GKPDAAKKGVVKAEKSKKKKEEEEEDDEEDE ,EEDEDEEEDD DE 165 185 215 Figure 3.1. (A) Schematic representation of HMGB1 and its deleted polypeptides with their abbreviations. HMGB1 consists of two HMG-box domains, A and B (black boxes), lysine rich basic regions (white boxes), and a C-terminal acidic tail (gray box). Numbers represent residues of N- and C-terminals in each protein. (B) Sequence of duplex DNA probes and abbreviations. Boldface font denotes the cisplatin 1,2-intrastrand d(GpG) cross-link (N1 GGN2) and flanking sequences. (C) Full sequence of HMGB1 and sequence alignment of HMG domain proteins. The intercalating residues of HMG boxes are indicated by vertical boxes and the acidic C-terminal tail of HMGB1 is indicated by underline. 137 5 AB165 dom A _ _ HMGB1 ~-- dom B - c _ - C T C C T C T C A G G A T C TC T _ - " C C T C T 3' _ - -W 000 -_ - _ - _40. _ _ _ - - - _- _ - - -.. -,_ - _ak _ _ _ _ , _. * _ _ _^w, -_ _ -_ _- - _-W _-t -_ m _ _ Figure 3.2. Footprint analysis of the interaction between HMGB1 proteins and 35AGGA. 35AGGA (-13 nM) was incubated with dom A (0.12 and 0.24 M), AB185 (0.9 and 1.8 tlM), HMGB1 (4 and 8 M), or dom B (1.2 M). The binding mixtures were subjected to hydroxyl radicals and analyzed by 20% denaturing PAGE. The arrows indicate bands corresponding to the platinated 1,2-d(GpG) cross-link site. The protected regions by dom A, AB185, and HMGB1 are shown by solid lines. The dot line indicates the region protected by dom B. 4-. 138 HMGB1 A * --- ,r~400WOOO O ~ Aw" 14 B ** * AB165 I 4Wht, a* l- - ..a Figure 3.3. EMSA analysis of HMGB1 and AB165 binding to 25TGGA. (A) Increasing amounts of HMGB1 (10 nM to 8 gM,left to right) were mixed with radioactively labeled 25TGGA (1 nM) (B) Increasing amounts of AB165 (0.07nM to 6 nM, left to right) were mixed with radioactively labeled 25TGGA (0.1 nM). 139 A HMGB1 HMGB1 F102A 1121A HMGB1 F37A -W WMWFWW - B AB165 sg~,.6... AB165 F37A :-- mOIIIMO I _ _ , , "c wd~~-·Q~ II-YLY il-q AB165 F102A 1121A .. . -- cc - ]Figure 3.4. EMSA analysis of HMGB1 and AB165 mutants binding to 25TGGA. (A) Increasing amounts of HMGB1 mutant proteins (10 nM to 200 nM, left to right) were mixed with radioactively labeled 25TGGA (~1 nM) (B) Increasing amounts of AB165 mutants (3.3 nM to 260 nM, left to right) were mixed with radioactively labeled 25TGGA (0.1 nM) containing 50 ng of poly[dGdC] as competitor DNA. 140 _ - HMGB1 HMGB1 AB165 F37A I 1~ We4100 we, -0 .,111 -_ "_*4 " *--- 0* ***A. 410 t!.i-. _ 44_* . _W- ~ -W- " mO "Moo " mm NO a - F37A F37 * -. ABI65 - 1 -'" - _ - -dr * . - Figure 3.5. Footprint analysis of the interaction between HMGB1 mutant proteins and 35AGGA. 35AGGA (-13 nM) was incubated with HMGB1, HMGB1 F37A (2 and 6 AM), AB165, or AB165 F37A (0.13 and 0.39 iM). The binding mixtures were subjected to hydroxyl radicals and analyzed by 20% denaturing PAGE. The arrows indicate bands corresponding to the platinated 1,2-d(GpG) cross-link site. The protected regions by wild type HMGB1 and AB165 are shown by solid lines. Dot lines indicate the region protected by HMGB1 F37A and AB165 F37A. 141 A I,,\ ,ua CNBr Cleavage of HMGB1 io At 37 - 0AM**. domain A 1 13 52 6375 t t tt domain B C tail 132 215 25 1I- 15 1 D 2 3 t 4 - 26.6 *- +(2-13)... +(14-52) +(76-132) > (133-215) (133-215) + (76-132) (76-215) 16.9 (133-215) 14.4 . (64-132) -- (76-132) 6.5 F- - 3.5 (14-52) < 4 KDa I Figure 3.6. CNBr cleavage analysis of EDC cross-linked HMGB1. (A) HMGB1 (5 M) was treated with increased amounts of EDC (0, 1, 4, and 10 g, lanes 1-4) and separated by 15% SDS-PAGE. (B) EDC cross-linked HMGB1 samples, prepared by the same method as (A), were cleaved by CNBr and separated by 16.5% Tris-tricince PAGE. Brackets indicate bands corresponding to the cross-linked peptide fragments by EDC. (C) Schematic representation of CNBr cleavage sites in HMGB1. Arrows indicate 5 of 6 CNBr cleavage sites in HMGB1, excluding the N-terminal methionine. 142 HMGB1 A + HMGB1 KDa 25TGGA cc--1 / EDC ,--"7 37 25 *** IWOMMOOMMMM 15 12345 67 8 _CC B EDC -Complex -.-25TGGA 1 2 3 4 5 Figure 3.7. EDC cross-linking of HMGB1 bound to cisplatin-modified DNA. (A) HMGB1 (5 jM) was treated with increasing amounts of EDC (0, 1, 4, and 10 4g, left to right) without (lanes 1-4) or with 7 M of 25TGGA (lanes 5-8) and separated by 15% SDS-PAGE.The amount of cross-linked HMGB1 is computed based on band intensities quantitated by densitometry. For free HMGB1, the portion of cross-linked protein with varied amounts of EDC is 0, 42, 91, and 100% (lanes 1-4). For the HMGB1-25TGGA complex, the values are 0, 65, 100, and 100% (lanes 5-8). (B) HMGB1-25TGGA complexes were treated with EDC as described in Figure 3.7A (lanes 5-8) and applied to 10%native PAGE to separate the free DNA and protein DNA complex. Lane 1 contains free 25TGGA and lanes 2-5 contain the same samples as lanes 5-8 in Figure 3.7A. 143 A KDa No I II III EDC 37 25 15 B HMGB1_NoEDC HMGB1 EDCI 4Complex 4 HMGB1 EDCIl HMGB1 25TGGA EDCIII *Complex 4 25TGGA Figure 3.8. EMSA analysis of cross-linked HMGB1 samples binding to 25TGGA. (A) HMGB1 (5 /uM) was treated with increased amounts of EDC (0, 1, 4, and 10 Pg, depicted as No, I, II, and III, respectively) and separated by 15% SDS-PAGE. (B) Increasing amounts of cross-linked HMGB1 samples prepared from (A) (0.1 /PM to 4 M, left to right) were mixed with radioactively labeled 25TGGA (2 M) and applied to 10% native PAGE to separate the free DNA and protein DNA complex. 144 Chapter 4 Multiple States of Stalled T7 RNA Polymerase at DNA Lesions Generated by Platinum Anticancer Agents * The work in this chapter has been published in J. Biol. Chem.,2003, 278, 52084-52092. 145 Introduction The anti-cancer drug cisplatin [cis-DDP, cis-diaminedichloroplatinum(II)] and related platinum anticancer agents such as oxaliplatin [(1R,2R- diaminocyclohexane)oxalatoplatinum(II)] and carboplatin [cis-diammine (1,1'cyclobutanedicarboxylato)platinum(II)] are used successfully to treat various types of cancers, including those of the head, neck, and testis (1). Much evidence reveals that the cytotoxicity of these platinum compounds is related to their ability to attack cellular DNA. Platinum agents react with the N7 atoms of the purine bases, forming 1,2intrastrand and 1,3-intrastrand cross-links as the major adducts. These DNA adducts inhibit essential cellular processes including transcription and trigger cell death. The 'detailed mechanism by which the platinum drugs selectively kill cancer cells is a subject of continued study in our laboratory and many others. A better understanding of cellular responses to platinum-DNA lesions is being sought to develop more effective cancer therapies through the optimization of Pt-DNA chemistry in the context of biochemical responses to the cytotoxic lesions. Platinum-DNA adducts are encountered and processed by many cellular proteins, events that determine the ultimate outcome of DNA damage (2-5). Among these proteins, RNA polymerase has become a major focus of study because of its various roles in processing damaged DNA. Several types of DNA lesions, including cisplatin cross-links (6-11), inhibit transcription by blocking RNA polymerase (12-15). Arrested RNA polymerase not only functions as a damage recognition factor, eliciting transcription-coupled repair (TCR), but also triggers programmed cell death, or apoptosis (16,17). The dominant consequence of RNA polymerase blockage by platinum-DNA adducts, either damage repair or apoptosis, will strongly bias the fate of cancer cells treated with the drugs. 146 DNA adducts of cisplatin block a variety of RNA polymerases including those present in E. coli,bacteriophage SP6, T7, and mammals (6,10,11,18,19).Different kinds of cisplatin-DNA adducts, 1,2-intrastrand d(GpG) and 1,3-intrastrand d(GpNpG) crosslinks, provide different levels of transcription inhibition. In vitro transcription by RNA polymerase II in human cell extracts is strongly inhibited by a 1,3-intrastrand, but not a 1,2-intrastrand adduct, whereas both types of cross-link efficiently block T7 and SP6 RNA polymerases (6,19). To date, however, little is known about the properties of stalled RNA polymerase at the site of platinum damage. In the present study, we employed site-specifically platinated DNA templates immobilized on a solid support in order to analyze the effects of defined platinum lesions on transcription and to isolate and study the stalled elongation complexes. Cisplatin 1,2-intrastrand d(GpG) and 1,3-intrastrand d(GpTpG) cross-links strongly block T7 RNA polymerase. Promoter-independent transcription on immobilized DNA templates allowed for the investigation of the exact stop sites of RNA polymerase at a platinum lesion under many different conditions. In addition to cisplatin, [Pt(dach)C12], which forms the same DNA adducts as oxaliplatin, was examined to understand the influence of the spectator ligand on transcription inhibition (Figure 4.1). We also investigated the transcription system whereby another polymerase trails the polymerase stalled at a site of platinum damage. Finally, the ability of RNA polymerase to resume transcription after being arrested at a platinum-DNA lesion was examined by chemically removing the platinum adduct. Experimental Procedures Materials. Histidine-tagged T7 RNA polymerase was expressed in BL21 cells carrying plasmid pBH161 (kindly provided by W. T. McAllister) and purified as 147 described (20). Cisplatin was obtained from Johnson-Matthey. The [Pt(dach)Cl2] compound was prepared as previously described (21). T4 polynucleotide kinase and T4 DNA ligase were purchased from New England Biolabs, and Dynabeads M280 Streptavidin from DYNAL. MagneHis Ni-Particles and RNasin (RNase inhibitor) were obtained from Promega. Construction of Site-Specifically Platinated DNA Templates. Platinated oligonucleotides containing cisplatin and [Pt(dach)C12] intrastrand cross-links were synthesized as reported previously (21). A 145 base-pair (bp) DNA template (T7 145) containing a T7 promoter and a site-specific platinum lesion was prepared by lenzymatic ligation of five synthetic oligonucleotide fragments and a sixth containing a platinum intrastrand cross-link (Figure 4.1B). 5'-Phosphorylated DNA fragments were annealed, ligated, and gel-purified as described previously (22). The platinum damage site was located in the template strand of the duplex DNA probe and a biotin moiety was placed at the 3' end of fragment T7 T50. For promoter-independent transcription, an 86-bp DNA probe with a 9-nucleotide (nt) 3'-overhang carrying a platinum intrastrand cross-link was prepared by ligating four oligonucleotides (Figure 4.1C). R:NA 8 was purchased from Dharmacon. Cisplatin 1,2-intrastrand d(GpG), 1,3intrastrand d(GpTpG), [Pt(dach)C12]1,2-intrastrand, and 1,3-intrastrand cross-links are abbreviated in this report as GG Pt, GTG Pt, GG dach, and GTG dach, respectively. Complete sequences of all DNA and RNA oligonucleotides are shown in Table 4.1. Promoter-DependentIn Vitro Transcription.The biotin-labeled DNA templates (T7 145, Figure 4.1B)were immobilized onto Dynabeads following the protocol provided by t]he manufacturer. Immobilized DNA templates were pre-equilibrated with the transcription buffer (40 mM Tris pH 7.9, 6 mM MgCl2, 2 mM spermidine, and 10 mM NaCl). Transcription by T7 RNA polymerase was carried out by incubating 148 approximately 5 nM DNA template with an equimolar amount of T7 RNA polymerase, 1U/pL of the Rnase inhibitor RNasin, 10 mM DTT, 0.12 mM ATP, 0.12 mM GTP, 5 PM UTP, and 1 M [a 2P]UTP in transcription buffer at room temperature for 5 min. The transcription elongation complex pauses after the synthesis of a 14 nt RNA transcript (EC14, Figure 4.1B) due to the lack of CTP in the reaction solution. Magnetic beads containing transcription complexes were separated from the solution by placing reaction tubes in a magnet rack (DYNAL). The supernatant solution was removed by aspiration with a pipette. The transcription elongation complex was washed with cold transcription buffer three times and used for next transcription steps. Single-round transcription assays were performed without magnetic separation of elongation complexes from the reaction solution. NTPs (1 mM final concentration) and 2 g of heparin were added into the transcription solution containing the paused elongation complex (EC14) formed by 0.12 mM ATP, 0.12 mM GTP, 5 M UTP, and 1 M [a3 2 P]UTP. Transcribed RNAs were ethanol precipitated and analyzed on 6% urea polyacrylamide gels. The gels were dried and visualized by autoradiography. Promoter-Independent In Vitro Transcription. A 5'-phosphorylated deoxyoligonucleotide was annealed with RNA 8, labeled with 32 p PI T50 at the 5' end, by heating at 50 °C and cooling slowly to room temperature (Figure 4.1C). An 8 pmol portion of this annealed probe was mixed with an equimolar quantity of T7 RNA polymerase in a total volume of 20 l of the transcription buffer described above. Following a 15 min incubation at 30 °C, approximately 200 pmol of PI C59 was added to the binding solution and incubation was continued for an additional 10 min. This artificial mimic of an elongation complex containing the 8-nt RNA segment (EC RNA 8) was extended by 6 nt to form a more stable complex containing a 14-nt RNA segment 149 (EC RNA 14) by incubation with 10 M of ATP and GTP. EC RNA 14 was isolated from excess probe and NTP by immobilization onto MagneHis Ni beads, which bind the histidine tag of T7 RNA polymerase (Figure 4.1C). Approximately 3 pmol of EC RNA 14 was obtained by this method. Next, a 2 pmol quantity of the 86-bp DNA probe containing a specific platinum cross-link was added to the immobilized RNA14 (1 pmol) elongation complex in 10 p1of transcription buffer containing T4 DNA ligase (2.5 U), 0.5 mM ATP, and 1% PEG8000. The ligation reaction was carried out at 12 °C for 1 h. The resulting EC RNA 14 complexes on the 145 bp DNA probe (PI 145, Figure 4.1C) were washed extensively and transcription buffer containing the indicated concentrations of NTP was added to initiate transcription elongation. Multi-Round In Vitro Transcription.Promoter-dependent transcription was performed in vitro as discussed above. Elongation complexes paused 14-nt from the start site (EC14) were isolated from the reaction solution (Figure 4.1B). Upon the addition of NTPs, isolated elongation complexes proceeded and then stalled at the site of platination. Approximately 2 pmol of additional T7 RNA polymerase was added to 1 pmol of the immobilized DNA template containing these stalled elongation complexes. Following this new round of transcription for 5 min, the magnetic beads were separated from the reaction mixture. RNA transcripts from both the beads and the supernatant solution were isolated and analyzed. Platinum Removal by Cyanide Ion Treatment. Separated elongation complexes EC14 were obtained following promoter-dependent transcription on 145-bp DNA templates containing the GG dach adduct. T7 RNA polymerases stalled at the damage sites were prepared by addition of 100 pM NTP. An additional elongation complex was prepared at a position (EC40, Figure 4.1B) 40 nt from the start site by adding only ATP, CTP, and GTP. EC14, EC40, and the stalled polymerases were washed with cold transcription 150 buffer three times to remove NTPs. Chemical removal of platinum adducts from each elongation complex was accomplished by treatment with 0.1 or 0.2 M NaCN in transcription buffer at 37 C for 30 min (23). The elongation complex was removed from NaCN solution by the magnetic beads. After washing the elongation complex with cold transcription buffer, transcription was resumed for each of the complexes by addition of 100 pM NTPs. Results Promoter-DependentIn Vitro Transcriptionon PlatinatedTemplates.An immobilized 145-bp DNA probe containing a biotin moiety at the 3' end of the template strand, a T7 promoter, and a platinum intrastrand cross-link was constructed as indicated in Figure 4.1. T7 RNA polymerase was employed to investigate minimal transcription in vitro with the immobilized DNA probe. Figure 4.2 shows that T7 RNA polymerase is capable of generating RNA transcripts following initiation on the immobilized DNA templates. In the absence of CTP, a transcription elongation complex comprising 14 residues of transcribed RNA transcript (5'-GAAUU AAUGG GGAU-3') was expected to form (Figure 4.1). An RNA transcript of 14 residues is produced from this reaction (Figure 4.2). In addition, many short RNA transcripts containing fewer than 10 residues are produced by abortive transcription initiation. Non-specific RNA transcripts longer than the expected 14 residues are also observed. CTP contamination in [a32 P]UTPand false transcription initiation before the start site are the most likely sources of these longer transcripts. Isolation of the transcription elongation complex on streptavidin-coated magnetic beads, however, removes all these abortive initiation products and diminishes the amount of non-specific transcripts (compare lanes 1 vs. 2). Transcription elongation 151 is resumed by adding 0.5 mM of NTPs (lanes 3, 6, and 9), which extends the 14-residue RNA in an elongation complex to a 125-nt RNA run-off transcript along the undamaged DNA probe (lane 3). Elongation stops at both cisplatin damage sites, GG Pt and GTG Pt (lanes 6 and 9, respectively), however. T7 RNA polymerase appears to stall at several positions in the vicinity of the platinum adducts (lanes 6 and 9). Transcription Bypass Through Platinum Binding Sites. Single-round transcription was performed by heparin treatment without magnetic separation of the elongation complexes (EC14). These elongation complexes, as well as EC14 products isolated by the magnetic bead methodology for comparison, were subjected to a second round of elongation to investigate bypass through the platinum lesions. In these two different transcription systems, DNA templates containing [Pt(dach)C12]1,2- and 1,3-intrastrand cross-links were employed together with probes containing the analogous cisplatin cross-links. As revealed in Figure 4.3, the bands for run-off transcripts in single-round transcription (lanes S) are more intense compared to those for transcription from isolated EC14 complexes (lanes B). The relative abilities of the individual adducts to inhibit T7 RNA polymerase, however, are unchanged by the two different experimental procedures. GTG dach, with a sterically larger spectator ligand, most strongly blocks T7 RNA polymerase. The polymerase bypasses GG dach adducts most efficiently, however, despite the bulky ligand. Overall, platinum 1,3-d(GpTpG) adducts block the polymerase more efficiently than 1,2-d(GpG) adducts. Promoter-Independent In Vitro Transcription. In vitro transcription on the immobilized DNA template allows us to use a 'transcription walking' method to provide the exact positioning of the polymerase along the DNA probe. The promoterdependent transcription methodology, however, generates several populations of RNA transcripts, which most likely originate from non-specific transcription initiation 152 around the start site (data not shown and see above). Multiple stop sites in the vicinity of the platinum adduct, such as those observed in Figure 4.2, might therefore derive from such false initiation. In order to circumvent this problem, we employed a promoter-independent T7 RNA polymerase transcription assay by assembling an elongation complex to study the exact stop sites of the stalled polymerase (Figure 4.1C). The construction of similar elongation complexes of yeast RNA polymerase II and T7 RNA polymerase using RNA:DNA hybrids has been reported previously (24,25). RNA transcripts corresponding to RNA48 and RNA62 were produced from 'transcription walking' along the undamaged DNA probe (Figure 4.4A,lanes 1 and 3). In this experiment, the 5' end of the T-95 DNA fragment was also labeled with 32p in order to confirm the ligation between an assembled EC and the 86-bp DNA probe (Figure 4.1C). 145-mer DNA probes (PI 145) and unligated 95-mer DNA fragments (T-95) are visible in Figure 4.4A. It is noteworthy that 133-nt run-off RNA transcripts are not detectable in this experiment, indicating that most T7 RNA polymerases stall at the GG Pt and GTG Pt lesions on the template strand. At a low concentration of NTP, the polymerase stops before the cisplatin 1,2-intrastrand d(GpG) cross-link, producing 62 residues of RNA transcript (Figures 4.4B and 4.4C). High NTP concentration drives the polymerase to the damaged d(GpG) site. At a cisplatin 1,3-intrastrand d(GpTpG) cross-link, most of the T7 RNA polymerase reaches the first G site of d(GpTpG) adduct, even at a low concentration of NTPs. The polymerase is capable of proceeding to the T residue of d(GpTpG) at high NTP concentrations. 2 + DNA adducts was also Blockage of T7 RNA polymerase by {Pt(dach)} examined by this method. In spite of the relatively high ability of T7 RNA polymerase to bypass a GG dach adduct in promoter-dependent transcription (Figure 4.3), no 153 detectable run-off RNA transcripts were observed on DNA containing either GG dach or GTG dach lesions (data not shown). The specific sites of NTP-driven translocation of elongation complexes into cisplatin and [Pt(dach)C1 2] cross-links following transcription are depicted in Figure 4.5A. T7 RNA polymerase is able to proceed only to the first G residue of [Pt(dach)C1 2] d(GpG) and d(GpTpG) cross-links (Figures 4.5A and 4.5B). UTP-Specific Incorporation by T7 RNA Polymerase at the Site of a Cisplatin 1,2Intrastrandd(GpG) Cross-Link.We further studied nucleotide incorporation by T7 RNAP at the site of platinum damage under conditions of high NTP concentration, with the aim of determining whether a specific nucleotide might be responsible for this effect. T7 RNAP elongation complexes, stalled before the damage site at low NTP concentration (10 tiM) and containing 62-nt RNA, were subsequently treated when 1 mM of ATP, UTP, GTP, CTP, or all 4 NTPs. As shown in Figure 4.6A, T7 RNAP inserts two additional nucleotides at the site of a cisplatin 1,2-d(GpG) intrastrand cross-link only when 1 mM UTP is present in addition to 10 ,M NTP, forming 64-nt RNA transcripts. Interestingly, the elongation complex is not able to transcribe into the damage site at a high concentration (1 mM) of ATP, GTP, or even CTP, the correct nucleotide for the d(GpG) template. A fraction of T7 RNAP, stalled at the first G site of the cisplatin 1,3d(GpTpG) cross-link, incorporates a nucleotide opposite T in the template strand of this lesion after addition of 1 mM NTP (Figure 4.6A). These RNA products are observed only at 10 IM NTP with 1 mM ATP or UTP. In a parallel experiment, we removed the 10 pM NTP from the reaction solution after forming the elongation complexes stalled at the site of a cisplatin 1,2-d(GpG) crosslink under the conditions (10 pM NTP) described above. Upon addition of 1 mM of ATP, UTP, GTP, CTP, or all 4 NTPs, we observed that only 1 mM UTP could drive T7 RNAP transcription into the d(GpG) site (Figure 4.6B). The data also indicate that T7 154 RNAP readily incorporates the incorrect nucleotide (UTP), not the correct one (CTP), at the site of the cisplatin d(GpG) cross-link. Figure 4.6B also demonstrates the presence of RNA products shorter than 62 nt only when ATP, GTP, or CTP is added to stalled polymerase. This result would appear to indicate intrinsic cleavage of RNA transcripts by stalled T7 RNAP. Multi-Round In Vitro Transcription. Recently, the ability of a trailing RNA polymerase molecule to alleviate an arrested RNAP was reported when more than one RNA polymerase transcribes along the same DNA template (26). Multi-round transcription was performed as described in Experimental Procedures to study the effect of a trailing RNA polymerase on polymerase stalled at a platinum cross-link. Upon treatment with additional RNA polymerase, the RNA transcripts in stalled elongation complexes are dissociated from the immobilized DNA template, whereas the transcripts remain at damage sites on the DNA templates without such treatment (Figure 4.7, lanes 5, 6 vs. 7, 8; lanes 9, 10 vs. 11, 12). T7 RNA polymerase that initiates from the same T7 promoter and follows the stalled elongation complex is responsible for the dissociation of the stalled polymerase from immobilized DNA, since added polymerases have no effect on the stalled polymerase in the absence of NTPs (Figure 4.7, lanes 1, 2 vs. 3, 4). Restarting Transcription Following Platinum Removal by Cyanide Ion Treatment. Cyanide ion has been successfully used to remove platinum adducts from DNA as the 2 - complex (23,27). This method was used to dissociate platinum from [Pt(CN) 4] immobilized DNA in the presence of T7 RNA polymerase stalled at the damage site. Platinum adducts were abstracted most efficiently from the [Pt(dach)C12] 1,2-intrastrand cross-link under our cyanide ion treatment conditions (data not shown). As indicated in Figure 4.8, most elongation complexes stay on the DNA templates after exposure to 155 NaCN. Upon treatment with cyanide ion, greater than 90% of the early elongation complexes EC14 are still active (lanes 2 and 4). On the other hand, 40% of EC40s are unable to resume transcription after 0.2 M NaCN treatment, and 0.1 M NaCN inactivates 30% of these complexes (lanes 6 and 8). A similar level of elongation complex inactivation occurred upon 0.2 M NaCl treatment, without any platinum abstraction (data not shown). These results are presumably the consequence of increased ionic strength. In the absence of cyanide ion, 16% of T7 RNAP bypasses the GG dach adduct, as indicated in Figure 4.3. Approximately 77% and 95% of active elongation complexes generate run-off RNA transcripts beyond the site of the platinum lesion after 0.1 M and 0.2 M NaCN treatment, respectively (lanes 2, 4, 6, and 8), indicating successful removal of the GG dach adduct. Similar levels of run-off transcripts are observed following re-elongation of stalled elongation complexes compared to EC40s following platinum complexation by NaCN. This result indicates that T7 RNA polymerase stalled at a platinum lesion is able to resume transcription after the damage is removed. Discussion Promoter-Dependentand -Independent Transcription Inhibition at Platinum CrossLinks. The transcription system in which platinum-DNA adducts are immobilized on a solid support provides a powerful tool to investigate the molecular mechanism of transcription inhibition by, and the properties of RNA polymerases stalled at, the major cross-links formed by cisplatin, carboplatin, and oxaliplatin. The DNA template attached to a solid phase provides a simple way to separate the transcription elongation complex from the reaction solution, allowing us to manipulate the position of the polymerase and detect the release of RNA transcripts from the complex. The activity of 156 the protein Mfd on stalled E. coli RNA polymerase was studied by using an immobilized DNA template in a similar strategy (28). The present results indicate that bacteriophage T7 RNA polymerase, a 100 kDa monomer, transcribes RNA from a biotinylated DNA template attached to streptavidincoated magnetic beads. Early elongation complexes (EC14, Figure 4.1), formed under CTP deprivation conditions, are fully capable of performing further transcription by subsequent addition of all 4 NTPs. The 14-residue RNA transcript in EC14 is labeled with [a32P]UTP(1 M), and incorporation of additional [a32P]UTP is prevented by isolating EC14 from the reaction solution on the beads and washing the immobilized elongation complex. Comparison of the relative amount of radioactivity in run-off (125 nt) versus platinum-aborted (-54 nt) transcripts, therefore, allows us to quantitate the extent of bypass compared to the amount of stalled T7 RNAP (Figure 4.1A). We also performed single-round transcription assays without isolation of EC14 from the reaction solution. In these experiments, EC14 formed in the presence of ATP, GTP, and [c32P]UTP (1 jiM) is elongated by adding 1 mM NTPs such that further incorporation of [a32 P]UTP is prevented due to the excess of unlabeled UTP in the reaction solution. As shown in Figure 4.3, these conditions afford more intense bands corresponding to run-off transcripts than observed for transcription from EC14 isolated on the magnetic beads. One possible explanation for this difference is that [a32P]UTP(1 puM)might be incorporated into RNA transcripts even in the presence of excess cold UTP (1 mM), which would produce a more intense signal for the longer (125 nt) run-off RNA products compared to the shorter (-54 nt) transcripts. Another possibility is that elongation complexes isolated by separation on magnetic beads might behave differently from those that are not separated in this manner from the reaction solution. 157 The ability of platinum intrastrand cross-links to block transcription was also investigated in a promoter-independent assay. Here, elongation complexes are assembled by addition of T7 RNA polymerase to a DNA:RNA hybrid. Almost no detectable run-off transcripts are observed for any of the four kinds of platinum adducts employed in this study (Figure 4.4). Thus the ability of T7 RNA polymerase to bypass platinum-DNA adducts following transcription from elongation complexes varies according to the methodology employed to investigate them. While this manuscript was in preparation, a report appeared that also demonstrated T7 RNA polymerase blockage at a single cisplatin adduct, with 70% and 90% efficiency of transcription inhibition by cisplatin 1,3- and 1,2-intrastrand cross-links, respectively (11). The various in vitro transcription systems applied to study transcription inhibition by platinum lesions (6,10,11),including the present one, clearly demonstrate that the major adducts of the anticancer drugs can significantly affect the process. The ability of different RNA polymerases to bypass a lesion is manifest, for example, by in vitro RNA polymerase II transcription initiated in human cell extracts, which transcribes efficiently through a cisplatin 1,2-intrastrand cross-link (6), compared to transcription by rat liver RNA polymerase II with purified transcription factors, which is strongly inhibited by the same adduct (11). Further work is required to determine the degree to which these findings reflect transcription inhibition by platinum-DNA adducts formed by the anticancer drugs in context of human cancer. A Closer Look at Polymerase Blockageby Platinum Adducts. T7 RNA polymerase obstruction by platinum adducts was investigated at the level of single nucleotide resolution by using the promoter-independent transcription system. T7 RNA polymerase stops just before a cis-diammineplatinum 1,2-intrastrand d(GpG) cross-link (Figure 4.4). At a high concentration of NTPs, however, the polymerase can add 158 additional nucleotides. RNA polymerases exist in multiple conformation states during transcription elongation (29). Several lines of evidence indicate that there are two active elongation states, fast and slow, and that the equilibrium between these two states is altered by the NTP concentration (30). RNA polymerase in the fast elongation state at high NTP concentrations might be able to drive RNA transcription up to the site of the platinum atom. Similar effect of NTP concentration on polymerase blockage by the analogous platinum 1,3-intrastrand d(GpTpG) cross-link also occurs (Figure 4.4). Although many bulky DNA lesions impede RNA polymerases, it is presently unclear exactly how the polymerases are stalled at the sites of damage. No structural information about an RNA polymerase arrested at a DNA adduct is yet available. A recent crystal structure determination of a T7 RNA polymerase elongation complex reveals the conformation of undamaged DNA at the active site (31,32). As depicted in Figure 4.9A, the nucleotide (+1) serving as template for the incoming NTP is severely distorted from the rest of the template strand. Upon incorporation of the nascent NTP at position +1, further elongation requires distortion of the +2 nucleotide from the +3 nucleotide (Figure 4.9A). Covalent cross-linking of the nucleotides at +2 and +3 by cisplatin, however, would prevent this distortion (Figure 4.9B). This information explains why the T7 RNA polymerase stops before the cisplatin 1,2-d(GpG) cross-link. Highly active polymerases can proceed to the +3 position, however, and different damage-induced overall conformations of the template strand at the active site will affect this process. In the case of transcription inhibition by a cisplatin 1,3-intrastrand d(GpTpG) cross-link (Figure 4.9, +1 and +3 nucleotides), T7 RNA polymerase is able to incorporate a NTP at the first G site of the adduct, even at low NTP concentrations (Figures 4.4 and 4.5). 159 In addition to the effects that structurally different cisplatin 1,2- and 1,3- intrastrand cross-links have on T7 RNA polymerase inhibition, our study of transcription using DNA containing [Pt(dach)C12]adducts reveals that the nature of the spectator ligands can also influence the process. T7 RNA polymerase, even in its highly active state, reaches only the first G residues of d(GpG) and d(GpTpG) adducts generated by [Pt(dach)C12](Figure 4.5). Besides preventing the structural rearrangement of the template strand required for transcription by covalent bonding, the bulky dach ligand might physically block further translocation of T7 RNA polymerase. At a low concentration of NTPs, the T7 RNAP elongation complex stalls just before the d(GpG) site and generates 62 residues of RNA, with U at its 3' end. Theoretically C is the next correct nucleobase to be added opposite the G site of the d(GpG) lesion (Figure 4.6C). A recent kinetic study revealed an allosteric effect of template-specific NTP binding to E. coli RNA polymerase, which shifts the equilibrium in favor of the fast elongation state (33). Based on this result, we anticipated that a high concentration of CTP might facilitate NTP incorporation by T7 RNAP at the template GG site of a cisplatin 1,2-d(GpG) cross-link. Unexpectedly, however, the elongation complex adds nucleotides at the damage site only when 1 mM UTP is employed (Figure 4.6). None of the other three nucleotides can affect such transcription elongation. The single-component T7 RNAP thus appears to be activated at high NTP concentrations in a different manner than the multi-component E. coli RNA polymerase. It is not clear at this point how a high concentration of UTP would activate the stalled T7 RNAP. It is likely, however, that the severely distorted GG template at a cisplatin 1,2-d(GpG) crosslink will not provide the geometrically correct template site for an incoming CTP (Figure 4.9). Thus even large amounts of CTP cannot activate the polymerase stalled at the damage site. UTP incorporation at a cisplatin 1,2-d(GpG) lesion inserts the incorrect 160 nucleotide into the transcript when T7 RNA polymerase bypasses the damage site. Several biological consequences, such as aborted or mutant transcripts, will ensure. Many RNA polymerases in both prokaryotes and eukaryotes are able to cleave their transcripts (34-36).Although such an activity has also been reported for T7 RNAP, little is known about the process (37). Such intrinsic transcript cleavage by T7 RNAP stalled at a damage site is observed in the present study (Figure 4.6B). Transcripts shortened by one or two nucleotides are generated by T7 RNAP, and this cleavage is not detected in the absence of NTP. Such nuclease activity requires a nucleotide that cannot be incorporated into the cisplatin d(GpG) damage site, namely ATP, GTP, or CTP. These nucleotides are not needed to provide energy to drive the cleavage reaction, since no cleavage occurs in the presence of 1 mM dATP (data not shown). We suggest that the activity we observe might reflect proofreading of transcription by this singlecomponent polymerase. More detailed study of nontemplated NTP-specific transcript cleavage by T7 RNAP is clearly warranted. The Effect of Multiple Polymerases at the Site of a Platinum-DNA Cross-Link. The recent discovery of cooperative transcription elongation by multiple RNA polymerases suggests a possible explanation for the observation of rapid RNA synthesis in vivo compared to single-round in vitro transcription (26). When more than one RNA polymerase molecule transcribes along the same DNA strand, the trailing polymerases appear to push forward and rescue the leading polymerases, which become active for transcription, from natural blocks such as pauses and arrests. In a follow-up study, it was reported that the trailing polymerases also rescue a polymerase from the roadblock created by DNA-binding proteins (38). Unlike these natural pause and arrest sites, it is difficult to predict a priori whether the strong physical block of a platinum cross-link would be circumvented by cooperative transcription elongation of multiple 161 polymerases. Nonetheless, in the multi-round transcription assay performed here, the trailing T7 RNA polymerases readily removed a polymerase stalled at a platinum lesion (Figure 4.7), allowing transcription to be continued. Although T7 RNA polymerase was used in this study, we expect similar cooperative elongation at a platinum lesion to occur with other types of RNA polymerases because of the conserved mechanical action of these enzymes. The E. coli transcription repair coupling factor, Mfd protein, pushes forward and dissociates a stalled E. coli RNA polymerase from the DNA template in the absence of NTPs (28,39). The present results may be particularly valuable for understanding transcription inhibition on highly active genes, such as those for ribosomal RNA (rRNA). Numerous RNA polymerase I molecules are transcriptionally active on the rRNA gene, whereas only one RNA polymerase II is generally elongating on most genes in a human cell (4042). Preferential inhibition of rRNA synthesis by cisplatin occurs in vivo (43). For this highly active gene, the large number of bound RNA polymerase I molecules will assure that cisplatin adducts are continuously encountered. A leading RNA polymerase I stalled at a cisplatin cross-link will be displaced, ultimately affording its rescue or the abortion of RNA synthesis. An abundant nuclear protein, human transcription release factor 2 (HuF2), dissociates efficiently both RNA polymerases I and II stalled at a cyclobutane thymine dimer (44). The relative ability to displace RNA polymerases stalled at DNA lesions by nuclear proteins such as HuF2 or trailing polymerases must be addressed to understand fully how the cell responds to transcription blocked by damaged DNA. Resumption of Transcriptionof T7 RNA PolymeraseStalledat a Platinum Binding Site. Like the two states of active elongation complexes, stalled RNA polymerases also exist in multiple conformational states. A portion of the polymerases is backtracked and 162 some of them are completely inactivated (29). In our study, approximately 40% of T7 RNA polymerase elongation complexes, stalled either by NTP deprivation or at a platinum lesion, were unable to resume transcription after addition of 0.2 M NaCN (Figure 4.8). Higher salt treatment and longer incubation of stalled T7 RNA polymerases generally inactivated the enzyme (data not shown). The ability of cyanide ion to remove a [Pt(dach)C1 2 ] 1,2-intrastrand cross-link from stalled elongation complexes at short incubation times resulted in a high population of active ECs. A T7 RNA polymerase stalled by NTP deprivation is similar to one arrested at a platinum lesion with respect to inactivation by NaCN treatment. Under this experimental condition, active elongation complexes of T7 RNA polymerase resume transcription through sites from which the damage has been removed. This result suggests that a platinum-DNA cross-link does not irreversibly alter the transcriptional activity of T7 RNAP. The ability of mammalian RNA polymerase II to resume transcription through a site of repaired damage, however, would depend upon its post-translational modification state, including phosphorylation and ubiquitination. Recently, ubiquitination of human RNA polymerase II, induced by transcription inhibition following cisplatin treatment, was reported both in vivo and in vitro (45,46). Cyanide ion treatment, such as that used in the present study, would allow the effect of chemical removal of platinum adducts on transcription by RNA polymerase II to be investigated. Any correlation between the post-translational modification state of RNA polymerase II and its ability to bypass a repaired site of damage would provide valuable information about TCR. Conclusion 163 Platinum 1,2- and 1,3-intrastrand cross-links strongly inhibit transcription by T7 RNA polymerase. The present study reveals that the elongation activity of T7 RNAP, which varies with the choice and concentration of NTPs, determines the stop site of the polymerase at a platinum cross-link. A highly active elongation complex is able to transcribe into the site of the platinum lesion, where the incorrect nucleotide UTP, rather than the correct nucleotide CTP, is readily incorporated at a cisplatin 1,2-d(GpG) cross-link. The severely distorted GG template of a platinum d(GpG) adduct does not appear to be appropriately positioned to form a base-pair with the correct nucleotide C TP. Our discovery of UTP incorporation suggests that mRNA and subsequent protein mutations might occur in a cell when the polymerase bypasses cisplatin damage sites, which could contribute to the cytotoxic effects of the drug. Cleavage by T7 RNAP of the nascent transcript reported here under conditions where the added NTP is not incorporated suggests a novel proofreading function for the enzyme. 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The complete sequences of DNA and RNA fragments employed in this study. ·__ Abbreviation Probes __ __ T7 C-59 5'-ACGTAATACGACTCACTATAGGGTTAATGGGGATCCACGAAA GCGACACCGGCACAGGG-3' T7 T-50 5'-GGTGTCGCTTTCGTGGATCCCCATTAACCCTATAGTGAGTCG TATTACGT-3' C-86 5'-ATGTTGAGGGGAATTCCGGTGAACATGCCTTTTGATGGAGCA GTTTCCAAATACACTTTTGGTAGAATCTGCAGGTGGATATTGAT-3' T-18 5'-CCTCAACATCCCTGTGCC-3' T-14 5'-TTCACCG*G*AATTCC-3' C-86 GTG 5'-ATGTTGAGGGGAATCACGGTGAACATGCCTTTTGATGGAGCA GTTTCCAAATACACTTTTGGTAGAATCTGCAGGTGGATATTGAT-3' T-14 GTG 5'-TTCACCG*TG*ATTCC-3' PI C-59 5'-GCTAGAATTAATGGGGATCCGAAGGGCCAAGCAAAGACGAAA GCGACACCGGCACAGGG-3' PI T-50 5'-GGTGTCGCTTTCGTCTTTGCTTGGCCCTTCGGATCCCCATTA ATTCTAGC-3' RNA 8 5'-GGGGAUCC-3' -- C-86/T-14 and C-86 GTG/T-14 GTG were used to prepare templates containing platinum 1,2- and 1,3-intrastrand cross-links, respectively. Boldface indicates T7 promoter (T7 C-59), 1,2-d(GpG) (T-14), and 1,3-d(GpTpG) (T-14 GTG) sites. Asterisks denote the platinated nucleosides. 170 A HN\ HN/ H2 NPtO I \C, [Pt(dach)C]I HOxaliplatin Cisplatin B C-86 5' T7 promoter T7 C-59 5 3' - Biotin- - T7 T-50 [Pt(dach)CI2 Oxaliplatin T-18 T-14 T7 promoter - T7 145 RitiPt , Rintin- T-63 ] - - - - - - -54nt ---------------------- 125 nt run-off ----- ----- ----- ----- AGGGG AAUU-3' (54 nt) 5'-GGGUU AAUGG GGAU5'-GGGUU AAUGG GGAU- ----- GGCACAGGGA-3' (EC40) 5 ' -GGGUUAAUGGGGAU-3' (EC14) T7 Promoter-CCCAATTACC CCTAG GTGCT TTCGC TGTGG CCGTG TCCCT ACAAC TCCCC TTAA6&CCAC... L C ...TTAGxCCAC... C PI C-59 5' 3' PIT-50 % bead T-18 T-14 T7 pol I _ C-86 5' RNA 8 , · ~,,_~--- % _ - r T-63 3' 86 bp probe 3 I . 1,3 d(GpTpG) 3' C-86 5' · 1,2 d(GpG) T-ql, r bead "45' , , -' '3' ' 3 PI 145 -------------- …---62 nt -1--- - - -- - - -- -- -- - - -- -- -- - -133 -- nt run-off Figure 4.1. Construction of DNA templates. (A) Structures of cisplatin, oxaliplatin, and [Pt(dach)Cl1].(B) Scheme illustrating the preparation of DNA templates for promoterdependent transcription, indicating the locations of the biotin moiety, T7 promoter, and platinum adduct. Sequences of the template strand between the T7 promoter and platinum site are depicted together with the expected RNA products initiated from the T7 promoter. The complete sequence is given in Supporting Information. (C) Schematic representation of promoter-independent transcription. DNAs and RNAs are indicated by solid and dotted lines, respectively. Each fragment is named according to its length on the template (T) or coding (C) strand. 171 No Pt 1 2 GG Pt 3 GTG Pt 4 56 7 8 9 run-off 125 nt to "i -~-54 nt .,~,,, m11~` _, elli ~iil!1 a ri' -- 14 nt it Figure 4.2. Gel-electrophoresis analysis of promoter-dependent in vitro transcription on immobilized DNA templates containing cisplatin intrastrand cross-links. Transcription by T7 RNA polymerase was performed as described in the text with an undamaged D)NA template (No Pt, lanes 1-3) and templates containing cisplatin 1,2-intrastrand d(GpG) (GG Pt, lanes 4-6) and 1,3-intrastrand d(GpTpG) cross-links (GTG Pt, lanes 7-9). Elongation complexes containing 14 nt of transcripts were formed under CTP deprivation condition (lanes 1, 4, and 7). Immobilized DNA templates containing elongation complexes were separated from the reaction solutions (lanes 2, 5, and 8) and subsequently treated with 0.5 mM NTP (lanes 3, 6, and 9). RNA transcripts were analyzed on a 6% urea polyacrylamide gel. The sizes of the RNA transcripts were determined by 'transcription walking' experiments (data not shown). 172 GG Pt S B GTG Pt GG dach S S B B Om*000 GTG dach S B _ run-off - 125 nt ! ::t54 nt 53 nt 9 5 5 3 20 16 4 2 Bypass (%) Figure 4.3. Gel-electrophoresis analysis of transcription bypass through cisplatin- and oxaliplatin-DNA intrastrand cross-links. Elongation complexes containing 14-residue transcripts (EC14)were treated with 1 mM NTP and heparin to afford a single round of transcription (lanes marked S). In a separate experiment, EC14s were separated from the solution as described in the text and treated with 1 mM NTP (lanes marked B, for beads). RNA transcripts were analyzed on a 6% urea polyacrylamide gel. The percentages of run-off to total RNA products through GG Pt, GTG Pt, GG dach, and GTG dach adducts are given in the bottom of each lane in the gel. 173 A GG Pt No Pt GTG Pt -- 145mer DNA run-off 133 nt --95mer DNA RNA62 nt -, -_ _ " " _ " RNA48 nt --. B GG Pt 5 RNA 62 C 5'-GGG GAUCC 5'-GGG GAUCC 5'-GGG GAUCC ...TACCC CTAGG --------GAAGG CTTCC RT RT 37 RT 37 RT 250 50 5 250 50 37 GTG Pt 37 RT 37 37 [NTP] (M) Temperature ----- ----- ----- ----- ----- ----- ----- ----- AGGGG AAUU-3' (EC RNA62) ----- ----- ----- ----- ----- ----- AGGGA -3' (EC RNA48) G-3' (EC RNA14) CGGTT CGTTT CTGCT TTCGC TGTGG CCGTG TCCCT ACAAC TCCCC TTAAGGCCAC...1,2 d(GpG) ... TTAGTGCCAC...1,3 d(GpTpG) RNA62 nt Figure 4.4. Gel-electrophoresis analysis of promoter-independent transcription on templates containing a cisplatin 1,2-d(GpG) or 1,3-d(GpTpG) cross-link. Experiments were performed as described in the text. Transcription from EC RNA14 on undamaged, immobilized templates (No Pt) proceeded to EC RNA48 following addition of 50 PM of ATP, CTP, and GTP. EC RNA62 was produced by adding 50 ,M of ATP, GTP, and UTP to the EC RNA48 product after washing. A 50 M portion of NTP was used to generate 133 residues of run-off RNA transcripts from EC RNA14 using the No Pt template. The EC RNA14 complex formed on templates containing a platinum lesion (GG Pt and GTG Pt) were treated with the indicated amounts of NTPs at room temperature (RT) or 37 °C. (A) 8% urea polyacrylamide gel analysis of RNA products. (B) Enlarged portion of the gel data in (A) containing RNA transcripts of RNA EC62 and stalled elongation complexes at platinum damage sites. (C) Sequence of the template strand up to the damage site (bottom lines) and of the RNA transcripts generated (top three lines). 174 A GTG Pt GG Pt GTG dach GG dach RNA 62 [NTP] mo -40*0 Ntm - 00 000"~H *00us.fturn ''" 0, so* - B GG Pt ... TTAA6GCCAC... -ttt *- . . High [NTP] High [NTP] .TTAGTGCCAC... -*·---, High [NTP] GG dach . .. TTAAGGCCAC... -"------ GTG Pt GTG dach . . High [NTP]tt High [NIP] .TTAGTGCCAC... High [NTP] Figure 4.5. Analysis of T7 RNA polymerase blockage by various platinum adducts. (A) Promoter-independent transcription on DNA templates containing GG Pt, GTG Pt, GG dach, and GTG dach was examined as described in the text. EC RNA14 complexes on templates containing a platinum lesion were treated with 10 M, 100 PM, or 1 mM NTP at room temperature. RNA transcripts in RNA EC62 complex and stalled elongation complexes at platinum damage sites were analyzed on 8% urea polyacrylamide gels. (B) Schematic representation of T7 RNA polymerase stop sites in the vicinity of each platinum lesion. 175 With 10 M NTP A GG Pt RNA RNA 62 64 - ATP UTP GTP GTG Pt CTP NTPs - ATP UTP [~~~~~~~~~~~~~ B GTP CTP ____*a" C With no NTP -- -AAUU NTPs W 1 mM a 62nt NN GGPt . . . TTAAGGCCAC... GG Pt - ATP UTP GTP CTP VW IW"' NTPs 1 mM 62 nt 00 --- 64 nt WW -*- 62 nt - -AAUCN GTGPt ... TTAGTGCCAC... 62 nt Figure 4.6. Analysis of nucleotide incorporation by T7 RNA polymerase at the site of platinum cross-links. Promoter-independent transcription on DNA templates containing GG Pt or GTG Pt was examined as described in the text. EC RNA14 complexes on templates containing a platinum lesion were treated with 10 PM NTP at room temperature. (A) Polymerases stalled at platinum damage sites at 10 M NTP were additionally treated with 1 mM ATP, UTP, GTP, CTP, or all 4 NTPs. RNA 62 and RNA 64 markers were prepared by 'transcription walking' through undamaged template containing no platinum. (B) After 10 PM NTP was removed from the reaction solution, 1 mM ATP, UTP, GTP, CTP, or all 4 NTPs was added into polymerases stalled at platinum damage sites. The resulting RNA transcripts were analyzed on 8% urea polyacrylamide gels. (C) Schematic representation of nucleotide incorporation by T7 RNA polymerase at the site of each platinum lesion. 176 B S T7 Pol B S B B 1 mM NTP 1 mM NTP No NTP S+ S B B S B B S S B S B 9 10 11 S run-off 125 nt ii 54 nt 1 2 3 4 GG Pt 5 6 7 8 12 GTG Pt Figure 4.7. Gel-electrophoresis analysis of transcription by multiple polymerases on the DNA templates containing a cisplatin lesion. Elongation complexes stalled at a cisplatin 1,2-d(GpG) cross-link were isolated as described in the text and incubated at room temperature for 5 min with (lanes 3 and 4) or without (lanes 1 and 2) additional T7 RNA polymerase. The same reactions were performed in the presence of 1 mM of NTP (lanes 5-8). Stalled elongation complexes by a cisplatin 1,3-d(GpTpG) cross-link were also examined in the same way (lanes 9-12). RNA products remained on the beads (B) or in the solution (S) were separated and analyzed. 177 EC40 EC14 0.1 [NaCN] (M) DNA marker (bp) M S 0.2 B S 0.1 B 0.2 Stalled 0.1 EC 0.2 S B SB S B S B - 145mer DNA *- run-off 125 nt -- 54 nt 100 1W 480112 50 -- 40 nt 20 -1 2 3 4 5 6 7 8 14 nt 9 10 11 12 Figure 4.8. Gel-electrophoresis analysis of platinum removal from DNA in elongation complexes by cyanide ion treatment. Isolated EC14, EC40, and stalled elongation complexes (stalled EC) were treated with NaCN as described in the text. After the reaction, the elongation complexes were separated from solution using the magnetic beads. RNA products in the solution (S) were directly analyzed, whereas elongation complexes on the beads (B) were further treated with 100 gM NTP and analyzed on 6% urea polyacrylamide gels. 178 B A cisplatin 1,2-d(GpG) cisplatin 1,3-d(GpTpG) of,, + =2, / ((r B _ H Figure 4.9. (A) Structure of the template strand at the active site of a T7 RNA polymerase elongation complex (32). The templating nucleotide for the incoming NTP is indicated by +1. Upstream and downstream nucleotides are indicated by -N and +N, respectively. (B) Structures of cisplatin 1,2-intrastrand d(GpG) (47) and 1,3-intrastrand d(GpTpG) (48)cross-links. All structures were generated by using Swiss-PdbViewer. 179 Chapter 5 RNA Polymerase II Blockage by Platinum DNA Damage: Polyubiquitylation of Stalled Polymerase 180 Introduction The anti-tumor activity of platinum-based drugs is mediated by their ability to attack DNA. The resulting DNA lesions generate numerous cellular signals, which eventually decide the fate of cells treated by platinum agents (1,2). Our understanding of this complicated process requires knowledge of the proteins that interact at the site of platinum damage on DNA and alter the consequential outcome for the cell. Among these proteins, those that arrive first at the platinum-DNA lesion most likely have the greatest influence (3). The identification of these proteins and comprehension of their functions are therefore important for elucidating the cellular responses to platinumDNA damage. RNA polymerase (Pol) II is responsible for transcribing most eukaryotic genes. Pol II is one of the proteins that encounter platinum lesions at a relatively early stage in the DNA damage-response process. This conclusion is supported by the high abundance of Pol II, with -300,000 copies in a single cell (4). Moreover, a photobleaching experiment revealed that almost 25% of this enzyme is constantly transcribing cellular DNA (5). Platinum-DNA adducts inhibit transcription by physically blocking RNA polymerases, as extensively demonstrated by many laboratories including our own (6-8). Cisplatin 1,2- and 1,3-intrastrand cross-links almost completely block T7 RNA polymerase and human RNA polymerase II. However, information about the effects of platinum-damaged DNA on Pol II, and of the stalled polymerase on damage repair, is incomplete. There is considerable evidence that the arrested RNA polymerase II initiates transcription-coupled repair (TCR), ensuring more efficient restoration of actively transcribing DNA templates (9). Although the exact mechanism of TCR is still unclear, it is generally accepted that stalled Pol II must be removed or translocated from the site 181 of damage in order for the repair machinery to assemble (10). In vitro transcription experiments demonstrated that arrested Pol II at the site of cyclobutane pyrimidine dimers (CPD) on the template strand is fairly stable but is dissociated from the damage site in whole cell extracts, possibly by human transcription release factor 2 (HuF2) (11,12). A recent study also reported ATP-dependent release of Pol II stalled at a cisplatin lesion from the template strand in whole cell extracts (8). Despite numerous such in vitro studies, the outcome of Pol II blockage and the mechanism of subsequent TCR initiation are not fully understood. Polyubiquitylation of Pol II following DNA damage by ultraviolet (UV) radiation and cisplatin treatment has been reported (13,14).This modification, however, does not occur in cells lacking Cockayne syndrome (CS) proteins (CSA and CSB), which are essential for TCR. In vitro transcription experiments with damaged DNA also demonstrated that Pol II ubiquitylation occurs in a transcription-dependent manner, further suggesting a possible link between ubiquitylation of the polymerase and DNA repair (15). Although a yeast study revealed ubiquitylation-mediated Pol II degradation following DNA damage coordinated by Rad26 (CSBhomolog)-Defl complexes (16),the roles of Pol II ubiquitylation in human cells remain to be addressed. Polyubiquitin chains linked via Lys-48 signal proteosomal degradation of the target and are a major form of protein modification. Proteins are less frequently ubiquitylated by Lys-63 of ubiquitin, an event that triggers various pathways distinct from protein degradation (17). Beside Lys-48 and Lys-63, five other lysines are available to form polyubiquitin chains, with many of their roles yet to be determined. Recently, Lys-63-linked ubiquitylation of Pol II was reported when Pol II was inhibited by aamanitin in nuclear extracts obtained from cells synchronized in S phase (18). This 182 modification could have diverse roles in processing arrested Pol II in response to DNA damage. To explore the effects of cisplatin-DNA damage on RNA polymerase II, the nature of Pol II blockage by the drug was studied in both whole cells and cell extracts. First, site-specifically platinated DNA templates immobilized on a solid support were employed to investigate the properties of stalled Pol II elongation complexes. Both RNA transcripts and Pol II proteins in ternary complexes were analyzed. Next, Pol II ubiquitylation upon transcription-inhibitory DNA damage was examined in order to identify the specific modifications and their effects on the properties of Pol II. In vitro experiments were performed with a series of ubiquitin mutants containing different sets of lysine residues available for modification of chain formation. The nature of polyubiquitin chains attached to stalled Pol II was further studied in HeLa cells by expressing hemagglutinin (HA)-tagged ubiquitin and ubiquitin mutants. Here we demonstrate that lysines beside Lys-48 are also involved in Pol II ubiquitylation upon DNA damage in live cells. Experimental Procedures Materials. Cisplatin was obtained from Johnson-Matthey. The proteosomal inhibitor MG132 was purchased from Calbiochem. T4 polynucleotide kinase, T4 DNA ligase, and all restriction enzymes were procured from New England Biolabs. Dynabeads M280 Streptavidin was obtained from DYNAL. RNasin (RNase inhibitor) was purchased from Promega. His-tagged ubiquitin expression vectors (pQE30-HisUb, pQE30-HisK48RUb, and pQE30-HisK63RUb) were kindly provided by Dr. K. B. Lee and the proteins were prepared as previously described (18). His-tagged ubiquitin proteins containing only one lysine residue, with the other six lysines mutated to 183 arginines (His-K6onlyUb, His-K48onlyUb, and His-K63onlyUb), were purchased from Boston Biochem. HeLa cells were grown at 37 °C in a humidified atmosphere of 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. Constructionof DNA Templates.Plasmid pG5MLPG380 (a gift from Dr. K. B. Lee) containing an adenovirus major late (AdML) promoter was prepared from a 10 mL LB culture of XL1-blue cells harboring the plasmid by using a Promega midi-prep kit. The plasmid was further purified by 1% agarose gel electrophoresis to remove the nicked form of DNA and subsequently used for in vitro ubiquitylation assays. A linear DNA template consisting of an AdML promoter, 380 base-pair (bp) of an upstream fragment before the transcription start site, a site-specific platinum lesion on the template strand, and a biotin moiety at the 5' end of the template was constructed. A DNA fragment containing an AdML promoter was prepared by PCR amplification from plasmid pG5MLPG380 (Figure 5.1). A biotin moiety was placed at the 5'-end of the coding strand by performing the PCR reaction with a 5'-biotinylated primer. The recognition site (GGTCTC) of restriction enzyme Eco31I, which produces a nonpalindromic 4-nucleotide (nt) 5' overhang, is located near the downstream end of the PCR product. The excess primers, NTPs, and DNA polymerases were removed from the PCR product by passage through Sephadex G-50 spin columns, followed by phenol extraction. The resulting DNA was treated with Eco31I and purified on a 4% native polyacrylamide gel, producing the DNA fragment with a 4-nt 5' overhang (Figure 5.1; PCR_promoter). Single-stranded DNA containing a cisplatin 1,2- or 1,3-intrastrand cross-link was synthesized as reported previously (19). A 95-bp DNA fragment with a site-specific cisplatin lesion and a 4-nt 5' overhang was prepared by enzymatic ligation of 5 pieces of 184 synthetic oligonucleotides (Figure 5.1; 99-95 DNA) following the previously described method (7). The complete sequence of each oligonucleotide piece is given in Figure 5.2. Phosphorylated oligonucleotides were annealed, ligated by T4 DNA ligase, and purified by denaturing polyacrylamide gel electrophoresis. Extracted 95-nt and 99-nt single-stranded DNAs were re-annealed by heating to 90 °C and slowly cooling to room temperature. The resulting 95-bp DNA fragment with a 4-nt 5' overhang was ligated with the Eco31I-treated PCR product containing an AdML promoter (Figure 5.1). The final DNA templates were purified on a 3.5%native polyacrylamide gel. Preparationof HeLa Nuclear Extract. HeLa nuclear extracts were obtained from Promega or prepared from HeLa suspension cells (obtained from American Cell Culture Center) following a previously reported method (20). Hydroxyurea (750 FM)treated HeLa cells were obtained as described previously (15). Cells were suspended in five times the packed cell volume (pcv) of low salt buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, and 0.5 mM DTT) and incubated for 15 min on ice. The cell suspension was centrifuged for 5 min at 420 xg and the swollen cell pellet was resuspended again in twice the pcv of low salt buffer. Cells were disrupted by using a dounce homogenizer with a type B pestle for ten strokes. The crude nuclear pellet was obtained by centrifuging for 10 min at 10,000xg. Two-thirds the pcv of high salt buffer (20 mM HEPES pH 7.9, 1.5 mM MgCl2, 25% glycerol, 420 mM NaCl, 0.2 mM EDTA, 0.5 mM PMSF, and 0.5 mM DTT) was added to resuspend the pellet and the nuclei were homogenized again with the douncer. After incubation with rotation at 4 °C for 30 min, the nuclear extract was obtained by centrifuging at 12,000 xg for 5 min. The resulting nuclear extract was dialyzed against storage buffer (20 mM HEPES pH 7.9, 20% 185 glycerol, 100 mM KC1,0.2 mM EDTA, 0.5 mM PMSF, and 0.5 mM DTT) and stored at 80 °C. In Vitro Transcription Assays in a HeLa Nuclear Extract. Transcription by human RNA polymerase II was carried out by incubating 300-500 ng of DNA template with approximately 50 Etgof HeLa nuclear extract, 1 U/ DtLof RNase inhibitor RNasin, 10 mM DTT, 1 mM ATP, 0.1 mM GTP, 0.1 mM CTP, and 10 DM [ca-32 P]UTP in transcription buffer (12 mM HEPES pH 7.9, 12% glycerol, 60 mM KCl, 8 mM MgCl2, 0.12 mM EDTA, 0.3 mM PMSF, and 0.3 mM DTT) in a total volume of 25 FL. Standard incubation was for 60 min at 30 °C. The reaction was stopped by adding 180 DtLof stop buffer (0.3 M Tris pH 7.4, 0.3 M sodium acetate, 0.5% SDS, 2 mM EDTA, and 3 tg/mL tRNA). For solid-phase transcription, the DNA templates were immobilized onto magnetic beads coated with streptavidin (DYNAL) in accord with the manufacturer's instructions and incubated with HeLa extract in the transcription buffer described above for 30 min at 30 "C before adding NTPs. Transcription elongation was initiated with NTPs containing 10 JtM [a-32 P]UTP and continued for 25 min. Non-radioactive UTP was added to 0.1 mM final UTP concentration and the reaction was incubated for an additional 5 min. Stalled elongation complexes on immobilized templates were isolated with a magnetic separator. Ternary complexes were washed with the transcription buffer containing the indicated detergents and further incubated under various conditions. Cyanide ion treatment to remove platinum from stalled elongation complexes was performed as described previously (7). A 1 M sodium cyanide stock solution was prepared in 10 mM Tris buffer pH 8.2 and stored at -80 C before use. RNA transcripts were isolated by phenol extraction, followed by ethanol precipitation, and analyzed by urea polyacrylamide gels. 186 To follow Pol II proteins in stalled ternary complexes, solid phase transcription was performed as described above except that 0.1 mM UTP total was used instead of 10 FM [a-3 2P]UTP. Polymerases in the reaction supernatant and wash solutions from immobilized templates on beads were applied to a 7.5% SDS polyacrylamide gel, blotted onto a PVDF membrane and probed with mouse anti-Pol II H14 IgM (Covance). In Vitro Ubiquitylation Assays in a HeLa Nuclear Extract. Ubiquitylation assays with plasmid DNA were performed by following a previously reported method (15). A 50 tg portion of HeLa nuclear extract was incubated with -1 tg of DNA template in transcription buffer, with and without 5 tM a-amanitin, for 15 min at 30 °C. To initiate transcription elongation and subsequent ubiquitylation, NTPs (1 mM ATP, 0.2 mM each C/G/UTP) and 1 g of various His-tagged ubiquitin proteins were added to the reaction solution. Following 40 min of additional incubation, ubiquitylated proteins including Pol II were separated from the nuclear extract by incubating with 20 tL of NiNTA agarose resin (Novagen) in His-NTA binding buffer (50 mM sodium phosphate pH 7.9, 0.3 M NaCl, 10 mM imidazole, and 0.05% Tween 20) at 4 °C for 1 h. The resin was washed twice with the His-NTA binding buffer containing 30 mM imidazole before eluting ubiquitylated proteins by adding SDS-loading buffer (20 mM Tris pH 6.8, 10% glycerol, 100 mM 2-mercaptoethanol, 1% SDS, and 0.02% bromophenol blue) supplemented with 0.1 M EDTA. The eluted proteins were separated on a 7.5% SDS polyacrylamide gel and analyzed as described above with mouse anti-Pol II H14 IgM (Covance). The assay was also carried out with bead-immobilized linear DNA templates. The immobilized template (1 kg) was incubated with HeLa nuclear extract (50 g) as described above for 30 min at 30 C before adding NTPs (1 mM ATP, 0.2 mM 187 C/G/UTP). Transcription was inhibited either by addition of 5 M a-amanitin or by using a site-specifically platinated DNA template. Following a 10-min transcription elongation reaction, ubiquitylation was initiated by adding 1 tg of ubiquitin and continued for 40 min. The beads were separated and washed with detergents as described above. RNA polymerase II proteins in each sample were examined by Western blot analysis with mouse anti-Pol II H14 IgM (Covance). Cellular Protein Fractionation. HeLa cells were collected by a cell scraper or trypsinization, with or without cisplatin treatment. The obtained cells were sequentially extracted by various detergents and salts according to a previously reported method ('21).All solutions contained a protease inhibitor cocktail (Calbiochem). Cells harvested from a 100 mm dish (-5 x 106cells) were lysed with 500 tL of hypotonic buffer (10 mM HEPES pH 7.9, 10 mM KC1, 1.5 mM MgCl 2, and 0.1% Triton X-100) on ice for 10 min. The nuclear pellets were separated from the supernatant, designated as the S2 fraction containing cytoplasmic proteins, and washed with isotonic sucrose buffer (50 mM Tris pH 7.4, 0.25 M sucrose, and 5 mM MgC12), producing the STM fraction. The washed nuclear pellets were extracted with a series of low salt buffers (10 mM Tris pH 7.4 and 0.2 mM MgCl 2) supplemented with 1% Triton X-100, 0 M NaCl, 0.3 M NaC1, 0.5 M NaCl, or 2.0 M NaC1, yielding the TW, LS, 0.3, 0.5, or 2.0 fractions, respectively. Finally, the remaining nuclear residue was solublized in the low salt buffer by sonication (NR fraction). A 10 - 20 tL portion of each cell fraction was applied to SDS-PAGE and analyzed by immunoblotting with the following antibodies: mouse anti-Pol II H14 IgM (Covance), mouse anti-actin IgG (Upstate), rabbit anti-HMGB1 IgG (Upstate), mouse anti-histone H3 IgG (Upstate), and rabbit anti-TFIIH p89 IgG (Santa Cruz biotech.). 188 To examine the portion of transcriptionally engaged Pol II, cellular proteins were fractionated as described (22) with a minor modification. HeLa cells obtained from a 25 cm2 culture flask (-2 x 106cells) were suspended in 400 tL of cytoskeleton buffer (10 mM PIPES pH 6.8, 100 mM NaC1, 300 mM sucrose, 3 mM MgCl 2, 0.5% Triton X-100, 1 mM DTT, and 1 mM EGTA) and incubated on ice for 10 min. The supernatant (S fraction) was separated from the nuclei, which were subsequently extracted with 400 tL of TD buffer (10 mM sodium phosphate pH 7.2, 150 mM NaC1, 0.5% Triton X-100, 0.5% sodium deoxycholate, 5 mM EDTA, and 2 mM EGTA) to yield the NE fraction. The remaining nuclear residue was boiled in 100 tL of hot SDS buffer (10 mM sodium phosphate pH 7.2, 150 mM NaCL, 1% SDS, 5 mM EDTA, and 2 mM EGTA) and diluted with 300 btLof cold TD buffer. The solution was sonicated to disrupt chromatin further and centrifuged to produce the NR fraction. HA-tagged Ubiquitin Expression in HeLa Cells. Plasmid MT127 (named as MTHA_Ubxl in this report; shown in Figure 5.3), comprising a CMV promoter and a HA-tagged ubiquitin gene, was obtained from the Bohmann laboratory (23). Site- directed mutagenesis was performed to mutate the indicated lysine residues of ubiquitin to arginines. The expression vectors (Figure 5.3; MTHA_Ubxn), which produce mRNAs encoding precursor proteins consisting of multimeric ubiquitins and ubiquitin mutants (n = number of ubiquitin in multimer), were constructed as described in Figure 5.3. The ubiquitin gene contains a BsmAI recognition site at its 3' end. BsmAI digestion affords a non-palindromic 4-nucleotide (nt) 5' overhang, essential for generating a repeated gene segment without the reverse gene sequence. An additional BsmAI site was introduced at the 5' end side of an HA-Ub gene by PCR amplification using primers P2 and P3, where P2 is 5'- CCAGGAGGGTCTCTGAGGTGGGATGGC- 189 TAGCTACCCTTATGAC-3' and P3 is GATGCTATTGCTTTATTTGTAACC-3',to give PCR_B. The PCR product obtained from the reaction with primers P1 (5'GGCGGTAGGCGTGTACGGTG-3') and P3 has only one BsmAI site and a NotI site (PCRA). Ligation of BsmAI digested PCR_A with an excess amount of BsmAI digested PCR_Bproduced a series of linear DNA fragments containing multimeric HA-tagged ubiquitin genes (Figure 5.3). Ligation products were separated in a 1% agarose gel and the desired DNA fragment was excised from the gel. Purified linear DNA was ligased ,with Eco31I treated PCR_B,yielding DNA with repeated HA-Ub genes flanked by NotI and EcoRI restriction sites. The final product was digested with NotI and EcoRI and ligased into the same restriction sites in MTHA_Ubxl. All plasmids were verified by DNA sequencing. MTHA_Ubx7 containing a heptameric ubiquitin gene was used for the present study. HeLa cells were grown to 90% confluence on a 6-well plate. Approximately 2 big of plasmid DNA was transfected with 10 tL of Lipofectamine 2000 reagent (Invitrogen) following the manufacturer's protocol. After 24 h incubation with transfection complexes, the medium was replaced with standard DMEM medium. Cells were washed with cold PBS and harvested with trypsinization. HA-tagged ubiquitin expression was determined by Western blotting with rabbit anti-ubiquitin IgG (Calbiochem) and anti-HA tag IgG (Rockland). Immunoprecipitation. HeLa cells (90% confluent) in a 25 cm2 culture flask (2 x 106 cells) were transfected with 12 Fig of HA-tagged ubiquitin expression vector by using Lipofectamine 2000 reagent. Following 24 h of protein expression, cells were treated with cisplatin and collected with trypsinization. Total cell extracts were prepared by boiling the cells in 100 ptLof the hot SDS buffer and diluting in 600 FtLof the cold TD 190 buffer as described previously (24). The solution was sonicated and cleared by centrifugation. Obtained cell extracts were incubated with anti-HA antibody for 3 h at 4 °C and proteins were captured by protein G-plus agarose beads (Calbiochem). The beads were washed five times with wash buffer (hot SDS buffer: TD buffer = 1: 6). Proteins were eluted from the beads by SDS loading buffer and analyzed by a Western blotting with mouse anti-Pol II H14 IgM (Covance). Immunoprecipitations of cell fractions with anti-HA antibody were carried out by diluting each fraction solution in six times volume of TD buffer, adding antibody, and capturing proteins as described above. Results Stability of RNA Polymerase II Stalled at a Platinum-DNA Lesion. A linear DNA template containing an AdML promoter and a cisplatin-DNA lesion with a biotin moiety was prepared (Figure 5.1). In vitro transcription assays were carried out with the probe. The undamaged probe (No Pt) is expected to produce a run-off (RO)transcript of 185 nt. On the other hand, RNA transcripts approximately 114 nt in length will be generated when a cisplatin lesion stops transcription elongation as illustrated in Figure 5.1. [a-32 P]UTPwas incorporated into RNA products during in vitro transcription. The undamaged probe (No Pt) yielded the expected RO transcripts from the transcription reaction (Figure 5.4A; lane 1). Transcription with damaged probes (GG Pt and GTG Pt) produced shortened RNA transcripts without any detectable RO transcripts, as shown in Figure 5.4A (lanes 2 and 3). The results indicate that cisplatin 1,2- and 1,3-intrastrand cross-links effectively block RNA polymerase II elongation in the present in vitro system. 191 Transcription assays were also performed with DNA templates immobilized onto streptavidin-coated magnetic beads. Pre-initiation complexes were formed on the promoter site by incubating HeLa extract with the immobilized templates. Transcription elongation is started by adding NTPs to pre-initiation complexes. Elongation complexes on the immobilized DNA templates were separated from the reaction solution at a different time point. RNA transcripts on beads (B) or in solution (S) were isolated and analyzed as shown in Figure 5.4B. As indicated above, a cisplatin 1,3-intrastrand cross-link strongly blocked the polymerase and yielded RNA transcripts -114 nt in length. Interestingly, most 114-nt transcripts were found on the beads (Figure 5.4B; lanes 2 and 4), indicating that RNA polymerase II stalled at a cisplatin lesion is fairly stable under this condition. Stalled elongation complexes appeared to remain on DNA templates for at least 2 h (data not shown). Detergent such as Sarkosyl and heparin have been successfully used in in vitro transcription assays to remove transcription initiation and unstable elongation complexes (25). Elongation complexes stalled at the site of cisplatin damage on the immobilized template were washed with transcription buffer containing 0.1 % Triton X100, followed by washing under more stringent conditions with solutions containing either 1% Sarkosyl or 100 Etg/mL heparin. As shown in Figure 5.4C, RNA transcripts in stalled ternary complexes were not eluted with these washes (lanes 4 and 6) indicating that the complexes are stable under these conditions. Stalled ternary complexes at the damage sites on immobilized DNA templates were also tracked by probing the Pol II protein. After solid phase in vitro transcription, the beads containing transcription complexes were separated from the reaction solution and sequentially washed with buffer solutions containing 0.1% Triton X-100, 1% Sarkosyl, and 2 M NaCl. Pol II in each fraction was analyzed with a Western blotting as 192 shown in Figure 5.5A. Only a small portion of Pol II was associated with the immobilized templates, which is typical for the in vitro transcription reaction with nuclear extracts. In the reactions with both undamaged and damaged probes, similar amounts of Pol II proteins were eluted by a 1% Sarkosyl wash (lanes 3 and 7), where proteins are possibly from transcription initiation complexes. Importantly, however, only in the presence of cisplatin adducts did a considerable amount of Pol II remain on the DNA templates, even after washing with 1% Sarkosyl (Figure 5.5A; lanes 4 versus 8). The data suggest that there are stalled Pol II proteins at damage sites that are resistant to a 1% Sarkosyl wash. To investigate the stability of Pol II stalled on the damage sites in cells, changes in the cellular and subnuclear distribution of Pol II upon DNA damage were examined. Following a reported procedure (21), proteins in HeLa cells were fractionated and analyzed with Western blots (Figure 5.5B). Most actin proteins were located in the cytosol whereas histone H3 proteins, strongly associated with chromatin, were found only in 2.0 and NR fractions (Figure 5.5B; left panel). TFIIH (p89) and HMGB1 proteins in the nuclei were extracted with a solution containing 0.3 M NaCl, indicating that the proteins were loosely bound to chromatin. The phospho-serine 5 version of Pol II (the elongating form of RNA polymerase II) was detected by antibody H14. Since the protein actively transcribes along chromatin, most proteins are non-extractable or extracted with only 2.0 M NaCl. Cells treated with cisplatin were also fractionated (Figure 5.5B; right panel). Upon DNA damage, TFIIH in the nuclei, initially detected mostly in the 0.3 fraction, is located in the 0.5, 2.0, and NR fractions, supporting the conclusion that, as a nucleotide excision repair (NER) component, TFIIH proteins tightly interact with chromatin after DNA damage. Cisplatin treatment also alters the 193 Pol II distribution. More Pol II was found in the NR fraction of damaged cells than NR fraction of non-damaged cells, as shown in Figure 5.5B (lanes 2.0 and NR). Dynamic State of Stalled RNA Polymerase II: Backtracking and Transcription Resumption. To study further the properties of stalled Pol II under different conditions, ternary complexes on immobilized templates were isolated and incubated with fresh nuclear extract. As shown in Figure 5.6A, most RNA transcripts remained associated with the immobilized templates on the beads, showing good stability of stalled Pol II under these incubation conditions. In the absence of NTPs, freshly added nuclear extract triggered RNA transcript cleavage (Figure 5.6A; lane 10). The data are consistent with previously reported polymerase backtracking and transcript cleavage induced by TFIIS (6,8). In the presence of both NTPs and nuclear extract only -114 nt RNA transcripts were obtained (Figure 5.6A; lane 8). The result suggests that, following polymerase backtracking and RNA cleavage, Pol II is still transcriptionally active and will resume elongation with NTPs until re-encountering the platinum-DNA lesion. Since stalled Pol II showed great stability at the site of cisplatin damage under our various experimental conditions, it was of interest to study whether stalled Pol II would be able to resume transcription beyond the DNA adduct after the damage is removed. In our previous study, cyanide ion was successfully used to complex platinum away from adducts on immobilized DNA in the presence of T7 RNA pclymerase (7). DNA templates containing cisplatin 1,3-intrastrand cross-links were incubated with 0.1 M NaCN (pH 8.2) for 30 min at 30 C and used for in vitro transcription. As shown in Figure 5.6B, 185 nt-long run-off transcripts were produced with damaged DNA templates only when the probe was pre-treated with cyanide ion (lanes 3 and 4 versus 5 and 6), indicating that a portion of cisplatin 1,3-intrastrand crosslinks was removed. Next, stalled ternary complexes were isolated and allowed to react 194 with cyanide ion in the presence of 1 mM NTPs. The 0.1 M NaCN treatment facilitated the release of more transcripts (-114 nt) in stalled elongation complexes from the templates (lanes 7 and 8 versus 9 and 10). The cyanide reaction, however, also provoked the generation of run-off transcripts as shown in Figure 5.6B (lane 7 versus 9). Platinum removal from stalled elongation complexes yielded similar levels of run-off transcripts (185 nt) when compared to transcription products (185 nt) of NaCN pre-treated DNA template (Figure 5.6B; lanes 5 and 6 versus 9 and 10). Taken together, our results indicate that stalled Pol II proteins at platinum lesions are transcriptionally active in nuclear extracts and capable of resuming transcription after the damage site when platinum is removed. Polyubiquitylation of Stalled RNA Polymerase II: In Vitro Ubiquitylation. Polyubiquitylation of Pol II upon transcription inhibition was reported in an in vitro system where nuclear extracts were obtained from synchronized cells in S phase (15). Following this work, Lys-63-linked polyubiquitylation of Pol II by a-amanitin inhibition was also demonstrated (18). To characterize further the nature of polyubiquitin chains on inhibited Pol II, we first investigated the ubiquitylation process by performing previously established in vitro assays with additional ubiquitin mutants. Nuclear extracts prepared from HeLa cells synchronized in S phase by hydroxyurea previously effected a-amanitin-induced ubiquitylation of Pol II (15). Similarly prepared HeLa nuclear extracts were incubated with plasmid DNA in the presence of ct-amanitin and His-tagged ubiquitin proteins. Transcription inhibition by a-amanitin was verified in a transcription assay (data not shown). After ubiquitylated proteins were collected by using a Ni-NTA resin, the level of Pol II ubiquitylation was analyzed by a Western blotting. The reactions with wild-type ubiquitin and both K48RUb and K63RUb 195 mutants generated clear stimulation of Pol II ubiquitylation (IIo-Ub) (Figure 5.7A; lane 3 versus 4, 5 versus 6, and 7 versus 8, respectively). Overall ubiquitylation of nuclear proteins by each mutant was examined with a Western blot detecting the His-tag epitopes (Figure 5.7B).By comparison to wild type and K63R ubiquitin, K48R ubiquitin afforded considerably less ubiquitylated protein. The data validate the conclusion that ubiquitylation of most proteins occurs via lysine 48 in our assays, which is consistent with previously reported results (18). His-tagged ubiquitin mutants containing only a single lysine residue (K48 only, K63 only, and K6 only) were also examined. As shown Iin Figure 5.7A, all mutants displayed increased levels of Pol II ubiquitylation (IIo-Ub) upon treatment of a-amanitin. Our data indicate that Pol II transcriptionally inhibited by a-amanitin can be polyubiquitylated by lysines other than Lys-48 in vitro under the present experimental conditions. Plasmid DNA was also globally damaged by cisplatin and examined for in vitro ubiquitylation. Although transcription was successfully inhibited by the resulting cisplatin lesions, stimulation of Pol II ubiquitylation with cisplatin-damaged plasmid was less apparent than in the experiment with ca-amanitin (data not shown). To study different properties of Pol II inhibited by ca-amanitin and cisplatin adducts, the ubiquitylation assay was carried out with immobilized linear DNA templates, where transcription was inhibited either by ca-amanitin or by using site-specifically platinated DNA templates. Following the ubiquitylation reactions, the beads containing transcription complexes were separated from the reaction solution (S) and sequentially washed with buffer solutions containing 1% Sarkosyl (W), and 2 M NaCl (E) (Figure 5.7C). As discussed in the previous experiment (Figure 5.5A), in an in vitro transcription assay most Pol II proteins are not associated with templates and therefore detected in 196 the reaction supernatant. To examine a relative level of Pol II ubiquitylation in each fraction, only one tenth of the total proteins in the reaction supernatant (S) and 1% Sarkosyl wash solution (W) were used for a Western blotting whereas all proteins in the 2 M NaCl elutant (E) were applied for a Western blotting (Figure 5.7C). Polymerases in the supernatant were ubiquitylated to similar levels in all the reactions as shown in Figure 5.7C (IIo-Ub in lanes 1, 4, 7, and 10), indicating that this ubiquitylation is most likely independent of damage-specific transcription inhibition. Immobilized templates were washed with 1% Sarkosyl to elute initiation and unstable elongation complexes as described above. Although comparable amounts of Pol II were washed by 1% Sarkosyl from all samples, ubiquitylated Pol II (IIo-Ub) was detected only in the presence of aamanitin regardless of the presence of cisplatin adducts (lanes 2 and 8 versus 5 and 11). Damage-specific Pol II proteins, which were tightly bound to DNA and therefore remained on the beads after a 1% Sarkosyl wash, appeared only in the transcription reaction with cisplatin-damaged templates in the absence of ca-amanitin (lanes 3, 6, and 12 versus lane 9). The level of ubiquitylation of these damage-specific polymerases, however, was relatively low compared to that of polymerases stalled by a-amanitin (IIo-Ub in lane 9 versus 11). Polyubiquitylationof Stalled RNA PolymeraseII in HeLa Cells. Previously, ubiquitin expression vector producing a precursor protein consisting of eight ubiquitin molecules was successfully used to express recombinant ubiquitin in HeLa cells (23). The precursor protein is endogenously processed into active ubiquitin proteins. In order to express HA-tagged ubiquitin and ubiquitin mutants in mammalian cells, plasmid vectors containing multimeric ubiquitin genes were constructed. Precursor expression and processing into HA-tagged ubiquitin was examined in HeLa cells transfected with 197 the resulting plasmids. Following the transfection, the expression of ubiquitin and ubiquitylated proteins was determined by immunoblotting with anti-HA epitope and anti-ubiquitin antibodies, as illustrated in Figure 5.8A. HA-tagged ubiquitin and ubiquitin mutants were successfully expressed in HeLa cells. The expressed protein level of HA-tagged ubiquitin, however, is relatively low compared to that of endogenous ubiquitin as examined with anti-ubiquitin antibody (Figure 5.8A; lanes 1 and 2). To determine the nature of polyubiquitin chains conjugated on Pol II inhibited by DNA damage in cells, HeLa cells were transfected with the plasmid vectors expressing a series of HA-tagged ubiquitin mutants and treated with cisplatin. Cellular proteins modified by HA-tagged ubiquitin were immunoprecipitated by using anti-HA tag antibody. As shown in Figure 5.8B, immunoprecipitation amplified the population of ubiquitylated Pol II (IIo-Ub in lane 1 versus 3). Cisplatin treatment is responsible for Pol :[I ubiquitylation since polyubiquitylated Pol II proteins were not detected in the absence of DNA damage (IIo-Ub in lane 3 versus 4). Three HA-tagged ubiquitin mutants, (K48R,K63R, and K48+63R),were expressed in HeLa cells. Following cisplatin treatment and immunoprecipitation with anti-HA tag antibody, similar levels of Pol II ubiquitylated by HA-ubiquitin were observed in cells expressing all three mutants as compared to those with HA-labeled wild type ubiquitin-expressing cells (Figure 5.8B; lane 3 versus lanes 5, 6, and 7). The result suggests that lysines other than Lys-48might be involved in the polyubiquitin chain formation on Pol II following DNA damage. To provide additional evidence for this idea, we tested two other ubiquitin mutants, K48only, containing only Lys-48 available for the chain formation, and NoK, containing no lysines for the process. As shown in Figure 5.8C, low levels of polyubiquitylated Pol II were detected upon cisplatin treatment in cells with K48only and NoK HA-ubiquitin 198 mutants compared with cells with other mutants (IIo-Ub in lanes 1, 2, and 3 versus 4 and 5). HeLa cells used in the present study were treated with cisplatin in the absence or presence of the proteosomal inhibitor MG132. The amount of phosphorylated forms (IIo) of Pol II clearly decreased following DNA damage, as shown in Figure 5.9. By adding MG132, however, the Pol II level was maintained over 10 h after DNA damage and even more ubiquitylated Pol II proteins (IIo-Ub) were observed. The data are consistent with previous work reporting that DNA damage induces down-regulation of Pol II following ubiquitylation of the protein (14). Several studies demonstrated that DNA damage-induced ubiquitylation occurs predominantly on the hyperphosphorylated form (IIo) of Pol II, which is transcriptionally engaged (26,27).Our understanding of the fate of ubiquitylated Pol II, however, is very limited. Here the stability of ubiquitylated Pol II was investigated in HeLa cells by observing only the modified form of the protein. HeLa cells were transfected with the HA-ubiquitin expression vector. Following protein expression and cisplatin treatment, cells were lysed with 0.5% Triton X-100 and centrifuged to afford cytoplasmic and soluble nuclear proteins (S fraction) (22). The pellet was subsequently treated with 0.5% Triton X-100 and 0.5% sodium deoxycholate to extract nuclear proteins loosely bound to chromatin (NE fraction). Based on our previous experiment (Figure 5.5B), Western blot analysis of TFIIH validated successful cell fractionation (Figure 5.10A; bottom panel). Approximately 40% of Pol II proteins were extracted by two detergent extractions (top panel; lanes 1 and 2 versus lane 3; lanes 4 and 5 versus lane 6). The ubiquitylated form (IIo-Ub) of Pol II, however, was mostly observed in NR fractions, suggesting that ubiquitylated polymerases are strongly bound to chromatin. 199 The proteosomal inhibitor MG132 prevents Pol II degradation following ubiquitylation upon DNA damage, resulting in accumulation of the ubiquitylated form of Pol II (Figure 5.9). HeLa cells were transfected and incubated with MG132 before cisplatin treatment. Pol II proteins were again fractionated and the ubiquitylated proteins were immunoprecipitated with anti-HA tag antibody. Similar levels of ubiquitylated Pol II were observed in NR fractions regardless of MG132 treatment as shown in Figure 5.10B (IIo-Ub in lanes 3 and 6). In S and NE fractions, however, significantly more ubiquitylated Pol II proteins were detected when the cells were treated with MG132 (lanes 1 and 2 versus 4 and 5). The results indicate that, in the absence of protein degradation, the accumulation of ubiquitylated Pol II occurs in the S and NE fractions. Discussion Stability of RNA PolymeraseII Stalled at a Platinum DNA Lesion. Previous work demonstrated ATP-dependent release of stalled Pol II on a cisplatin or a CPD lesion from the template in whole cell extracts (8,11). In these studies, early elongation complexes were first allowed to form from cell extracts or purified transcription factors. These isolated Pol II complexes were then further elongated to encounter the DNA adducts and subsequently incubated in cell extracts. In the present experiments, transcription elongation and subsequent polymerase blockage were performed in nuclear extracts without isolating early elongation complexes (Figure 5.4). Under these conditions, stalled Pol II proteins mostly remained bound to the DNA templates even in the presence of ATP and nuclear extracts as shown in Figures 5.4B and 5.4C. A possible explanation for the difference between these results and the previous Pol II release from the damage site would be a different level of release factor proteins, including HuF2 200 (12), available for processing stalled Pol II. When cell extracts are added to isolated elongation complexes, release factors supplied by this procedure will primarily work on these isolated complexes. In the present transcription reaction, which did not employed isolated Pol II complexes, however, fewer release factors would be available to process stalled Pol II at the damage site since they would have to attend to other transcription complexes. Moreover, when stalled Pol II proteins were separated from the transcription reaction solution and re-incubated with nuclear extracts in the presence of ATP (Figure 5.6), the extent of Pol II release varied from batch to batch of added nuclear extracts (data not shown). Some nuclear extracts displayed more Pol II release than others, probably because of different levels of release factors in each nuclear extract. Furthermore, cell fractionation experiments showed an increased level of chromatinassociated Pol II proteins following DNA damage (Figure 5.5B). The data suggest that at least some population of polymerases remains on the damage sites without being disrupted by release factors in living cells. At present, how cells decide to release stalled Pol II is yet to be determined. The nature of the lesion, overall DNA damage level, sequence context and gene identity at the site of inhibition, stage of the cell cycle, and local chromatin structure are only some of the possibilities that must be considered to address this question. Dynamic State of Stalled RNA Polymerase II: Backtracking and Transcription Resumption.Ternary complexes stalled at damage sites, such as cisplatin or CPD lesions, are targets for transcription factor TFIIS-mediated transcript cleavage (6,8,28). Under our experimental conditions, most stalled Pol II proteins remained on the DNA template with -114 nt RNA transcripts in the presence of nuclear proteins and NTPs, as discussed above. All RNA transcripts of these ternary complexes, however, were cleaved and became shorter when they were incubated with nuclear proteins in the 201 absence of NTPs (Figure 5.6A). Stalled Pol II that is not removed from the damage site by release proteins seems to be in a dynamic state, whereby the polymerase constantly backtracks, cleaves its RNA transcript with the help of TFIIS, and resumes elongation only to encounter the DNA lesion again. Platinum removal experiments on stalled ternary complexes further support the dynamic behavior of stalled Pol II (Figure 5.6B). Previously, CPD adducts were removed from the DNA template by using photolyase and light (28).In that study, some of the transcripts in ternary complexes stalled at CPD sites were elongated beyond the lesion following photolyase treatment. In the present 2study, 0.1 M NaCN (pH 8.2) was used to remove platinum adducts as [Pt(CN) 4] complexes; higher concentrations of cyanide ion significantly destabilized stalled Pol II complexes (data not shown). Although the reaction removed only a small fraction of 1,3 intrastrand cross-links on DNA templates, most Pol II proteins stalled at the platinum lesions resumed elongation past the damage sites as soon as the adducts were removed (Figure 5.6B). Previous in vitro studies performed with cisplatin and UV-damaged DNA (8,11) demonstrated that the presence of Pol II stalled at the damage site does not affect dual incision of the lesion by nucleotide excision repair factors. These results can be explained by the model discussed above whereby stalled polymerases are able to backtrack from the damage sites without leaving the DNA templates, these exposing DNA damage sites for dual incision by repair enzymes. Polyubiquitylationof Stalled RNA PolymeraseII. In response to DNA damage such as UV radiation and platinum anticancer agents, RNA polymerase II is ubiquitylated. Several studies in eukaryotic systems suggest a link between this event and DNA repair (13,16). The reason why Pol II becomes ubiquitylated upon DNA damage in human cells, however, is not fully understood. In the present in vitro study, transcription inhibition by a-amanitin stimulated Pol II ubiquitylation in nuclear extracts obtained 202 from hydroxyurea-treated HeLa cells, as reported previously (15). Stimulation of Pol II ubiquitylation does not occur when transcription is inhibited by cisplatin damage, however (data not shown). In the present work we performed ubiquitylation assays on immobilized templates to demonstrate that Pol II stalled by a-amanitin is eluted by a 1% Sarkosyl wash, whereas polymerase stalled by a cisplatin lesion is much more stable (Figure 5.7C). The data clearly reveal different stabilities of Pol II proteins when arrested by different methods. The ubiquitylated form of Pol II is observed only when the polymerases were halted by a-amanitin and not by cisplatin adducts on the immobilized linear DNA probes. It is possible that the fraction of Pol II stalled at the sites of cisplatin cross-links was too low for ubiquitin ligases to modify, whereas more Pol II proteins were inhibited by pre-incubation with a-amanitin. A recent in vitro study reported Pol II ubiquitylation via Lys-63 upon transcription inhibition by a-amanitin in nuclear extracts prepared from synchronized cells in S phase (18). Lys-63-linked polyubiquitylation of Pol II raises the intriguing possibility that the process not only triggers Pol II degradation but also sends additional, non-degradative signals in response to DNA damage. In the present study, stimulated ubiquitylation of Pol II induced by ca-amanitin occurred via Lys-6, Lys-63, and Lys-48 of the ubiquitin protein (Figure 5.7A). The nature of polyubiquitin chains formed on Pol II following cisplatin treatment was investigated in HeLa cells. Epitope- tagged ubiquitin and ubiquitin mutants were previously used to study the ubiquitylation of various target proteins (29). Consistent with our in vitro results, the data indicate that lysines other than Lys-48 are also involved in ubiquitylation of Pol II in response to DNA damage by cisplatin. Although the ubiquitin ligases responsible for ubiquitylation of stalled Pol II have not been identified, two proteins, BRCA1/BARD1 203 (27,30) and the von Hippel-Lindau (VHL) protein (31), are reported to ubiquitylate Pol II in vitro, as well as in cells, in a DNA damage-dependent manner. Both proteins specifically interact and target the hyperphosphorylated form of Pol II. Several studies suggested that BRCA1/BARD1 mediates ubiquitylation through lysine residues other than Lys-48 (32,33). In one case there was Lys-6-linked ubiquitylation by BRCA1/BARD1, which did not trigger degradation but rather affected the stability and activity of the target protein (34). It was suggested that there is more than one ubiquitin ligase that targets Pol II upon DNA damage in vivo (27,30).In response to DNA damage, Pol II proteins may be ubiquitylated by various ligases with a series of different polyubiquitin chains depending on the environment of stalled Pol II. A task for the future is to understand the consequences of these modifications on Pol II. Polyubiquitylation of Stalled RNA Polymerase II: Effect on Pol II. UV-induced ubiquitylation and subsequent proteosomal degradation of Pol II were previously reported (14). The present study also demonstrates that cisplatin treatment facilitates Pol II degradation following ubiquitylation (Figure 5.9). Moreover, the proteosomal inhibitor MG132 prevented the degradation and accumulated the ubiquitylated form of Pol II. It is plausible that Pol II degradation is one of the main cellular consequences of its blockage at DNA adducts. The exact reason for such Pol II degradation, however, is still unclear. One hypothesis is that Pol II degradation removes the polymerase from the DNA lesion so that the repair machinery can assemble. Several studies (14,35), including the present work (Figure 5.10A), demonstrate that ubiquitylated Pol II is tightly bound to chromosomal DNA, suggesting that even its ubiquitylated form is transcriptionally engaged. Despite different levels of DNA damage, and consequential levels of ubiquitylation, most ubiquitylated Pol II proteins are tightly associated with chromosomal DNA (Figure 5.10A). Upon the inhibition of proteolysis with MG132, 204 ubiquitylated Pol II accumulates as discussed above. We show here that this additional portion of ubiquitylated Pol II is exclusively unbound or at best loosely bound to DNA (Figure 5.10B).The levels of chromatin-associated ubiquitylated Pol II are unchanged by MG132 treatment. Under our experimental conditions, where cells were treated with lethal concentrations of cisplatin for a short period of time, we are most likely observing direct responses of Pol II to cisplatin DNA damage. The results suggest that a fraction of ubiquitylated Pol II is dissociated from the damage sites and rapidly degraded by proteosomes. The data also show that more ubiquitylated Pol II proteins remain tightly associated with chromatin than are dissociated following ubiquitylation in the presence of MG132 (Figure 5.10B). It remains to be determined how cells regulate the release of ubiquitylated Pol II from the damage sites, leading to prompt degradation of the protein. Conclusion The consequences of RNA polymerase II blockage by cisplatin lesions were studied outside as well as within living cells. From previous reports (8,11) and the current study, we portray in Figure 5.11 some of the possible biological consequences when Pol II encounters DNA damage. Upon Pol II obstruction by DNA damage, the polymerase can be removed from the damage site by cellular release factors including HuF2 (12). The process not only can expose the damage site for the global genome repair (GGR) of NER but also can rescue the stalled polymerase from destruction (Figure 5.11). Our data also indicate that a considerable level of stalled Pol II proteins can remain strongly associated with damaged DNA. These polymerases backtrack from the damage sites, cleave the transcripts, and re-elongate. This dynamic process is probably mediated by multiple proteins, including CSB and TFIIS (8). NER might take 205 place at the DNA damage site with Pol II remaining on DNA but backtracking from the lesion (11). Stalled Pol II can also be ubiquitylated by numerous ubiquitin ligases, conjugating polyubiquitin chains on the polymerase through Lys-6, Lys-48, Lys-63, and possibly other lysines of ubiquitin. Some portion of ubiquitylated Pol II will be released from DNA and rapidly degraded by proteosomes, while others will remain on DNA and may trigger non-degradative signals or affect the properties of stalled Pol II. Acknowledgments I would like to thank Professor P. A. Sharp for experimental guidance and helpful discussions. I also thank Cindy Yuan for assistance with constructing DNA templates. This work was supported by a grant CA 34992 from the National Cancer Institute. 206 References 1. Jamieson, 2. Wang, D., and Lippard, S. J. (2005) Nat. Rev. Drug Discov. 4, 307-320 3. Cline, S. D., and Hanawalt, P. C. (2003) Nat. Rev. Mol. Cell Biol. 4, 361-372 4. Kimura, H., Tao, Y., Roeder, R. G., and Cook, P. R. (1999) Mol. Cell. Biol. 19, 5383- E. R., and Lippard, S. J. (1999) Chem. Rev. 99, 2467-2498 5392 5. Kimura, H., Sugaya, K., and Cook, P. R. (2002) J. Cell Biol. 159, 777-782 6. Tornaletti, S., Patrick, S. M., Turchi, J. J., and Hanawalt, P. C. (2003) J. Biol. 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Nishikawa, H., Ooka, S., Sato, K., Arima, K., Okamoto, J., Klevit, R. E., Fukuda, M., and Ohta, T. (2004) J. Biol. Chem. 279, 3916-3924 35. Yang, L. Y., Jiang, H., Rangel, K. M., and Plunkett, 1495 W. (2003) Oncol. Rep. 10, 1489- 209 AdMLP Biotin Fr/AZ 5'-GCCC i GGGC-5' + 380 bp Pt 99-95 DNA PCR_promoter Biotin li, I rI- Pt _ - 114 nt RNA _ __ - - _ - - - - - - - - - - __ - - -18 nt RORNA Figure 5.1. Construction of the DNA template. The transcription probe was prepared by ligating two DNA fragments, PCR_promoter and 99-95 DNA as described in Experimental Procedures. PCR_promoter contains a DNA segment of 380 bp in length between the upstream end of the linear probe and the transcription start site (bent arrow) with a biotin moiety located at the 5' end of the coding strand (top strand). The sequence of each overhang is shown. Run-off (RO) and shortened transcripts, resulting from polymerase blockage by a cisplatin lesion (marked as Pt), are depicted as dotted lines with their expected sizes. 210 C-59 5'-GCCC C-40 99-95 DNA T-18 T-14 T-63 T-14GTG: TTCACCG*TG*ATTCC C-59GTG: CCCGGGCACAGGGATGTTGAGGGGAATCACGGTGAACATGCCTTTTGATGGAGCAGTTT T-14GG: TTCACCG*G*AATTCC C- 59GG: CCCGGGCACAGGGATGTTGAGGGGAATTCCGGTGAACATGCCTTTTGATGGAGCAGTTT '-63: ATCAATATCCACCTGCAGATTCTACCAAAAGTGTATTTGGAAACTGCTCCATCAAAAGGCATG Tr-18: CCTCAACATCCCTGTGCC C -40: CCAAATACACTTTTGGTAGAATCTGCAGGTGGATATTGAT Figure 5.2. The complete sequences of DNA fragments employed to construct 99-95 DNA. All sequences are depicted in the 5' to 3' direction. C-59 GG/T-14 GG and C-59 GTG/T-14 GTG were used to prepare templates containing platinum 1,2- and 1,3intrastrand cross-links, respectively. Boldface indicates 1,2-d(GpG) (T-14) and 1,3cl(GpTpG)(T-14 GTG) sites. Asterisks denote the platinated nucleosides. 211 P1 P ,IP2 BsrnAI HA-Ub CMV polyA --- MTHA Ubxl ( ~\, ~Notl ~I BsmAI EcoRI PCR_A (P1 & P3/ P3 "'~PCRB ,' (P2 & P3) Eco311 HA-Ub HA-Ub I I I BsmAldigestion I BsmAI EcoRI BsmAI BsmAI EcoRI Notl BsmA digestion Eco311digestion + Notl I ligation + ( Jn EcoRI Notl ligation| n I EcoRI Notl Cloning with Notl & EcoRI CMV MTHA Ubxn polyA HA-Ub n , II-- Figure 5.3. Schematic representation of expression vector construction for HA-tagged ubiquitin. Genes encoding HA-tagged ubiquitin is indicated with light gray boxes. The sequences of primers and detailed method are described in Experimental Procedures. 212 A B 10 ,,·0o0 C) I I S 30 B S Time (mn) B I *-185 S 1 2 I nt RO *-114 nt(Pt) 3 _ _-114 1 2 _as _N 3 I1 nt(Pt) 4 C S WT WS B WH B Ln 1 2 3 4 5 *-114 nt (Pt) 6 Figure 5.4. Gel electrophoresis analysis of in vitro transcription in HeLa nuclear extracts. Transcripts isolated from transcription reactions performed as described in Experimental Procedures were applied to 6% urea polyacrylamide gels. A, Transcription reactions were carried out in solution with the undamaged probe (No Pt; lane 1) and damaged probe containing a cisplatin 1,2-(GpG) or 1,3-(GpTpG) cross-link (GG Pt or GTG Pt, respectively). B, In vitro transcription on the immobilized DNA templates was performed as described in Experimental Procedures. Following addition of NTPs, the immobilized templates containing elongation complexes stalled at cisplatin I,2-(GpG) cross-links were isolated from the reaction solution at the indicated time points. RNA transcripts isolated from the beads (B) and the reaction supernatant (S) were analyzed. C, Immobilized probes containing stalled ternary complexes were isolated from the reaction supernatant (S) and washed with 0.1% Triton X-100 (WT). The resulting probes were washed with either 1% Sarkosyl (WS; lane 3) or 100 Etg/mL heparin (WH; lane 5). RNA transcripts isolated from the wash solutions and the remaining probes on the beads (B) after Sarkosyl and heparin washes (lanes 4 and 6, respectively) were analyzed. Run-off (RO) transcripts and RNAs (Pt) resulting from polymerase halting by cisplatin lesions are indicated. 213 A GTG Pt No Pt S wl w2 E S wl w2 E *-llo 1 B 2 3 4 5 6 7 8 Cisplatin treated No damage Chromatin bound Cytosolic S2 STM TW LS 0.3 0.5 2.0 NR H14 Cytosolic Chromatin bound I'. .. .. i S2 STM TW LS W en 4Own 0.3 0.5 2.0 NR H14 _ TFIIH(p89) TFIIH(p89) actin _c a - 1I HMGB1 Histone H3 I -H Figure 5.5. Western blot analysis of RNA polymerase II. A, Following solid phase in vitro transcription with the undamaged (No Pt) and cisplatin damaged (GTG Pt) DNA templates, the beads were separated from the reaction supernatant (S). The resulting beads were washed with 0.1% Triton X-100 (wl) and 1% Sarkosyl (w2), followed by eluting remaining proteins with 2 M NaCl (E). Proteins were applied to 7.5% SDSPAGE and immunoblotted with anti-Pol II antibody H14. The phospho-serine 5 version of Pol II (IIo) is indicated. B, Protein fractions were obtained from HeLa cells in the absence (left panel) or presence (right panel) of cisplatin treatment as described in Experimental Procedures. Each protein fraction, extracted from an equivalent number of cells, was subjected to 4-20% SDS-PAGE and immunoblotted with the indicated antibodies. 214 A _ + + - 1 mM NTPs _ + + Fresh NE wash SB S B SB S B 114 nt (Pt) 1 2 3 4 5 6 7 8 9 10 B + -+ No Pt S B + GTG Pt GTG Pt S B S B 0.1 M NaCN S B Fresh NE S B S B 4-185 I~~~~~~~~~~~~~A~ ,- Irrrrrll~~~~~ I nt (RO) . ~W ,: 4-1 14 nt (Pt) 1 2 3 4 5 6 7 8 9 10 11 12 Figure 5.6. Gel electrophoresis analysis of in vitro transcription in HeLa nuclear extract. A, Transcription complexes stalled at the sites of cisplatin 1,3-(GpTpG) cross-links were obtained and washed with 0.1% Triton X-100 and 1% Sarkosyl (lanes 1 and 2, respectively). Ternary complexes were further incubated in the transcription buffer (lanes 3 and 4) supplemented with NTPs (lanes 5 and 6), nuclear extract (lanes 9 and 10), or both (lanes 7 and 8) at 30 °C for 30 min. B, Transcription reactions were carried out with the undamaged (No Pt; lanes 1 and 2) and damaged probe containing a cisplatin 1,3-(GpTpG) cross-link (GTG Pt, lanes 3 and 4). The reaction was also performed with the GTG Pt probe pre-treated with 0.1 M NaCN at 30 °C for 30 min (lanes 5 and 6). Pol II proteins stalled at the damage sites were isolated and treated with 0.1 M cyanide ion with or without nuclear extract in the presence of NTPs. Following the reactions or incubation, the beads (B) were separated from the incubation solution (S) and transcripts were analyzed on 6% urea polyacrylamide gels. Run-off (RO) transcripts and RNAs (Pt) resulting from polymerase halting by cisplatin lesions are indicated. 215 - A No DNA WT + Ilo-Ub C Hi - WT + - 1 B 2 3 4 K48R K63R - - + w'! 5 7 6 C _t - K63 only + - K6 only + - 7. *J 4i.a 8 9 K63R - S _ V,"" 10 11 12 a-am 13 14 GTG Pt + E + .... No Pt K48R WT + · ," *. K48 only - SW E + a-am S W E SW E L 1 Ilo-Ub C 11o - 1 2 3 4 5 6 123 WB: anti-His Figure 5.7. Western blot analysis of in vitro ubiquitylation of RNA polymerase II. A, Plasmid pG5MLPG380 was incubated with various His-tagged ubiquitin proteins in HeLa nuclear extracts treated with 5 tM a-amanitin (a-am) for 15 min. Following 40 min of ubiquitylation reaction, ubiquitylated proteins including Pol II were collected by Ni-NTA resin. Proteins were applied to 7.5% SDS-PAGEand immunoblotted with antiPol II antibody H14. B, The pG5MLPG380 plasmid in HeLa nuclear extracts was incubated with His-tagged ubiquitin (His-Ub). a-Amanitin (a-am) was added to the reaction with wild-type His-Ub, His-K48RUb, and His-K63Ub (lanes 1, 2, and 3, respectively). Reaction mixtures were applied to 4-20% SDS-PAGE and blotted by an anti-His epitope antibody to detect the ubiquitylated proteins. C, Following solid phase in vitro ubiquitylation with undamaged (No Pt) and cisplatin damaged (GTG Pt) DNA templates in the presence and absence of a-amanitin, the beads were separated from the reaction supernatant (S). The resulting beads were washed with 1% Sarkosyl (W), followed by eluting the remaining proteins with 2 M NaCl (E). One tenth of solutions S and W and all of solution E were applied to 7.5% SDS-PAGE and immunoblotted with anti-Pol II antibody H14. The phospho-serine 5 version of Pol II (IIo) and polyubiquitylated Pol II (Ub-IIo) are indicated. 216 A B 1 2 3 45 6 Transfection Input 1 2 3 45 6 + + - + + + Cisplatin Ilo-Ub 110 1 2 3 4 5 6 C -\-A b;br > t8b-VO§Q SN + WB: Anti-HA 7 + + + + Transfection Cisplatin Ilo-Ub Anti-ubiquitin 1 2 3 4 5 Figure 5.8. Western blot analysis of ubiquitylation of RNA polymerase II in HeLa cells. A, Total cell extracts were obtained from HeLa cells transfected with control plasmid (lane 1), MTHA_Ubx7 (lane 2), MTHA_K48RUbx7 (lane 3), MTHA_K63RUbx7 (lane 4), MTHA_K48+63RUbx7 (lane 5), and no plasmid (lane 6). Proteins were subjected to 420% SDS-PAGE and immunoblotted with anti-HA tag and anti-ubiquitin antibodies. B and C, HeLa cells were transfected with the plasmid vectors expressing indicated HAtagged ubiquitin and ubiquitin mutants. Following cisplatin treatment (30 tM for 6 h) of these HeLa cells, ubiquitylated proteins were immunoprecipitated with anti-HA tag antibody as described in Experimental Procedures. Collected proteins were applied to 7'.5%SDS-PAGEand immunoblotted with anti-Pol II antibody H14. The phospho-serine 5 version of Pol II (IIo) and polyubiquitylated Pol II (Ub-IIo) are indicated. 217 5 tM MG132 + 1 3 6 10 1 3 6 10 300 AM Cisplatin incubation time (h) ] Ilo-Ub -lo 1 2 3 4 5 6 7 8 Figure 5.9. The effect of MG132 on ubiquitylation of RNA polymerase II in HeLa cells. HeLa cells were treated with cisplatin (300 FtM)in the presence and absence of MG132 (5 FiM)and collected at the indicated time points. Total cell extracts were obtained from the cells and applied to 7.5% SDS-PAGE and immunoblotted with anti-Pol II antibody H14. The phospho-serine are indicated. 5 version of Pol II (IIo) and polyubiquitylated Pol II (Ub-IIo) 218 A B 300 FtM,3h + 30 EM, 6h S 300 M, 2h NE NR S Cisplatin NE NR S NE NR S + Cisplatin + MG132 I NE NR ] Ilo-Ub -110o Input WB: H14 ] Input WB: H14 After IP with anti-HA ] o-Ub After IP -Io0 with anti-HA WB: H14 ] Ilo-Ub -li WB: H14 1 2 Input Ilo-Ub -110 3 4 5 6 WB: TFIIH 1 2 3 4 5 6 Figure 5.10. Subcellular localization of ubiquitylated RNA polymerase II in HeLa cells. A, HeLa cells were transfected with MTHA_Ubx7, treated with the indicated concentration of cisplatin (30 tM for 6 h and 300 tM for 2 h) and fractionated into three parts as described in Experimental Procedures. Each protein fraction, extracted from an equivalent number of cells, was subjected to 7.5% SDS-PAGE and immunoblotted with anti-Pol II antibody H14 before (Input) and after immunoprecipitation (After IP) with anti-HA tag antibody (top and middle panels, respectively). Input samples were also immunoblotted with anti-TFIIH antibody (bottom panel) B, Following transfection of HeLa cells with MTHA_Ubx7, the cells were treated with cisplatin (300 tM for 3 h) in the presence and absence of MG132 pre-treatment (5 FM). Cellular proteins were again fractionated and analyzed by a Western blotting before and after immunoprecipitation with anti-HA tag antibody (top and bottom panels, respectively). The phospho-serine 5 version of Pol II (IIo) and polyubiquitylated Pol II (Ub-IIo) are indicated. 219 [Pol II release & degradation XON Ski ,, , ,; ,, s _, _ _~'~ , GGR? GGR? ra >Cease factors t Pol II release] Stalled Pol II _ _ % t I TCR? 4- ,,_SB, '- fn0~~ Backtracking & re-elongation rl l- . Ubiquitin ligases TFIIS Non-degradative signals Figure 5.11. Model depicting the consequences of RNA polymerase II blockage by cisplatin adducts. Possible outcomes when Pol II is stalled at the site of DNA damage are shown. Two sub-pathways of nucleotide excision repair (NER), transcriptioncoupled repair (TCR) and global genome repair (GGR), are illustrated. RNA polymerase II (Pol II), DNA damage (Pt), RNA transcripts (dotted lines), and ubiquitin (Ub) protein are indicated. 220 Chapter 6 Following Cisplatin: Hapten-Conjugated Platinum Complex 221 Introduction For many years, studies on protein interactions with cisplatin-damaged DNA have been performed mostly with in vitro systems using synthetically prepared platinated DNA. Relative binding affinities and kinetic properties of damaged-DNA recognition proteins toward platinated DNA were studied. In a living cell, however, it is unclear which of these proteins respond and affect the cellular processes against DNA damage. Although there are numerous ways to track cellular proteins, strategies to follow DNA damaged by platinum agents in living cells are limited. Various hapten molecules such as fluorescein, biotin, and dinitrophenyl (DNP) moieties have been introduced into platinum compounds. Platinum complexes conjugated with a fluorescein or DNP were used to track platinum agents in a cultured cell (1). Recently, the dinuclear platinum complexes modified by carboxyfluorescein diacetae and dinitrophenyl were used for investigation of platinum cellular distribution (2). None of the reported complexes, however, were tested for their biological activity, and many of them were derived from biologically inactive forms of platinum compounds. In order to study the behavior of platinum anti-cancer drugs by using hapten-conjugated agents, it is vital to have novel platinum complexes which can mimic the biological activity of cisplatin. By introducing a hapten molecule such as biotin into a biologically active cisPt(II) compound, the drug in a cell can be monitored. Capturing the hapten molecule allows us to separate the damaged DNA from normal DNA, which is extracted from the cells treated with this compound. More importantly, this compound provides a novel tool to detect extremely low levels of the drug on DNA. This detection method might make it possible to study cellular protein interaction with cisplatin-induced DNA damage. 222 Platinum complexes containing a desthiobiotin moiety (DTB-Pt) with different tethers were synthesized. The complexes react with DNA in vitro and form lesions which can inhibit transcription and can also be recognized by HMGB1 protein. DNA damaged by DTB-Pt was fully recovered by using streptavidin-coated beads through the strong interaction between desthiobiotin and streptavidin. Moreover, a simple dot blot analysis detected less than 1 fmol of DTB-Pt DNA adduct. None of the complexes, however, are as cytotoxic as cisplatin in various cell lines. As an alternative strategy, an azide moiety was introduced into a platinum complex. Experimental Procedures Desthiobiotinamido-hexylamine-Boc solution of desthiobiotin (DTB6Nboc) (1) (Scheme 6.1). To a stirred (0.64 g, 3.0 mmol) and N-methylmorpholine 3.0 mmol) in 22 mL of dry N,N-dimethylforamide (NMM) (0.33 mL, (DMF) was added 6 mL of DMF solution containing N-Boc-1, 6-diaminohexane hydrochloride (0.75 g, 3.0 mmol) and NMM (0.33 mL). A solution of coupling reagent HATU (1.1 g, 3.0 mmol) in DMF (4 mL) was added to the reaction solution. The combined mixture was stirred at room temperature for 16 h, and DMF was removed by a rotary evaporator. Resulting yellow oil was purified on a silica gel column; elution with methanol (CH3OH )/dichloromethane (CH 2C1 2) = 1: 6 afforded 1.2 g (90%) of DTB6Nboc (1). RF, 0.75 (C'H3 OH/CH 2 C 2 = 1: 5). ESI-MS: m/e Calcd. (M + H +) 412.3, Found 412.3. 1H NMR (300 MHz, DMSO): b 7.72 (t, 1 H, amide NH), 6.79 (t, 1 H, amide NH-boc), 6.32 (s, 1 H, clesthiobiotin NH), 6.12 (s, 1 H, desthiobiotin NH), 3.60 (m, 1 H), 3.49 (m, 1 H), 2.98 (q, 2 H), 2.86 (m, 2 H), 2.00 (t, 2 H), 1.6 (m, 25 H), 0.95 (d, 3 H). Desthiobiotinamido-hexylamine(DTB6N) (2). 1 (1.2 g, 2.8 mmol) was suspended in 15 mL of ethyl acetate (EtOAc). After adding 5 mL of concentrated HC1, the reaction 223 solution was stirred for 30 min at room temperature. The mixture was dried under reduced pressure and redissolved in water (10 mL). The pH of the solution was adjusted to 12 with 2 M NaOH and water was removed to give a clear oil. The resulting DTB6N (2) was cleared from the inorganic salts by treating with methanol (4 mL) and discarding solids. Evaporation under reduced pressure affords 2 (1.2 g, >100%) containing small amount of salts. ESI-MS: m/e Calcd. (M + H+) 312.3, Found 312.3. H 7.75 (t, 1 H, amide NH), 6.38 (s, 1 H, desthiobiotin NH), 6.14 NMR (300 MHz, DMF): (s, 1 H, desthiobiotin NH), 3.74 (m, 1 H), 3.59 (m, 1 H), 2.98 (m, 2 H), 2.86 (m, 2 H), 2.20 (t, 2 H), 1.75 (m, 2 H), 1.6 (m, 14 H), 1.02 (d, 3 H). Diboc-aminoethyl-Gly (3). Boc-anhydride (2.4 g, 11 mmol) in 8 mL of methanol was added to a solution of aminoethyl-glycine (0.59 g, 5.0 mmol) and triethylamine (TEA) (2.5 mL, 16 mmol) in methanol (8 mL). The solution was stirred for 16 h at room temperature. After the removal of methanol, the crude mixture was purified by a silica gel column; elution with methanol! /chloroform (CHC13)= 1 : 4 containing 1%oacetic acid (AcOH) to (CH 3 OH/CHC1 give 1.35 3 /AcOH g (85%) of diboc-aminoethyl-gly (3). RF, 0.85 = 1 : 4: 1%). ESI-MS: m/e Calcd. (M + Na +) 341.4, Found 341.4. 1H NMR (300 MHz, CDC13 ): 6 5.22 (br d, 1 H, amide NH), 3.92 (br s, 2 H), 3.41 (br s, 2 H), 3.30 (br s, 2 H), 1.45 (s, 18 H). DTB9diamine (4). 3 (0.83 g, 2.6 mmol) and N-hydroxysuccinimide (NHS) (0.30 g, 2.6 mmol) were dissolved in 35 mL of dioxane. To the solution was added dicyclohexylcarbodiimide (DCC) (0.54 g, 2.6 mmol). After 4 h reaction at room temperature, the white precipitate was removed by filtration and NHS-activated 3 was partially purified by a silica gel column with 10% methanol in dichloromethane. Approximately 1 equiv of DTB6N (2) (0.82 g) and TEA (0.11 mL) in 15 ml of methanol 224 were added to a solution of NHS-activated 3. The reaction mixture was stirred at room temperature until completion, which was monitored by silica gel TLC analysis with CH3OH/CHC13 = 1 : 6. Following purification by silica gel chromatography, the resulting DTB9diamine-diboc compound was deprotected by 3M HCl as described above to give 4 (-0 74 g, 90%). ESI-MS: m/e Calcd. (M + H +) 412.3, Found 412.3. 1 H NMR (300 MHz, DMSO): 6 8.05 (t, 1 H, amide NH), 7.78 (s, 1 H, amide NH), 6.32 (s, 1 H, desthiobiotin NH), 6.14 (s, 1 H, desthiobiotin NH), 3.59 (m, 1 H), 3.48 (m, 1 H), 3.20 (m, 2 H), 3.05 (m, 4 H), 2.86 (m, 2 H), 2.69 (m, 2 H), 2.02 (t, 2 H) 1.6 (m, 14 H), 0.98 (d, 3 H). DTB9Pt (5). To a solution of 4 (0.23 g, 0.56 mmol) in water (3 mL) was added K2[PtCl 4] ( 0.21 g, 0.53 mmol) in water (2 mL). The reaction mixture was stirred overnight at room temperature. The yellow precipitate was collected, washed with cold water, and dried to yield 5 (90 mg, 23%). ESI-MS: m/e Calcd. (M + Na+) 701.3, Found '701.2. 1 H NMR (300 MHz, DMF): 6 8.18 (t, 1 H, amide NH), 7.70 (t, 1 H, amide NH), 6.21 (s, 1 H, desthiobiotin NH), 6.04 (s, 1 H, desthiobiotin NH), 5.20 (br s, 2 H, Pt-NH2), 4.19 (d, 1 H), 3.74 (m, 1 H), 3.62 (m, 1 H), 3.18 (m, 4 H), 2.98 (m, 1 H), 2.71 (m, 2 H), 2.46 (m, 1 H), 2.16 (t, 2 H), 1.6 (m, 16 H), 1.02 (d, 3 H). 195PtNMR (DMF): 6-2349. Cbz-6-amino-hexanol (6) (Scheme 6.2). Benzyl chloroformate (Cbz-Cl) (1.4 mL, 10 rnmol) was added to 15 mL methanol containing 6-amino-hexanol (1.2 g, 10 mmol) and TEA (1.4 mL, 10 mmol). The reaction mixture was stirred at room temperature for 16 h. The solution was dried and the crude product was purified by a silica gel column with CH 3OH/CH 2 C12 = 1 : 5 to yield 2.2 g ( 88%) of 6. H NMR (300 MHz, CDC1 3): 6 7.18 (s, 5 H), 5.11 (s, 2 H), 4.78 (br s, 1 H), 3.60 (t, 2 H), 3.19 (m, 2 H), 1.40 (m, 9 H). Cbz-6-amino-hexanal (7). Solid tetrapropylammonium perruthenate (TPAP) (nPr4N)RuO4 (70 mg, 0.2 mmol) was added in one portion to a stirred mixture of 6 (0.5 225 g, 2 mmol), N-methylmorpholine N-oxide (MMO) (0.35 g, 3 mmol), and 1 g of 3 A molecular sieves in distilled dichloromethane (8 mL) at room temperature under argon. The reaction followed by silica gel TLC (EtOAc/ CH 2C12 = 1: 2) was completed after 1 h. The mixture was filtered through a short pad of silica and eluted with ethyl acetate, yielding pure Cbz-6-amino-hexanal (7) (0.3 g, 60%). H NMR (300 MHz, CDC1 3): 9.79 (s, 1 H), 7.18 (s, 5 H), 5.14 (s, 2 H), 4.80 (br s, 1 H), 3.20 (m, 2 H), 2.41 (m, 2 H), 1.40 (m, 6 H). Cbz-6-amino-hexyl-diamine-diboc (8). 7 (0.29 g, 1.2 mmol) and boc-ethylenediamine (0.28 g, 1.7 mmol) were dissolved in 10 mL distilled methanol containing 100 PL acetic acid under argon. Sodium cyanotrihydroborate (0.1 g, 1.6 mmol) was added to the mixture portion wise over 45 min. The solution was stirred overnight at room temperature and cooled in an ice bath. A saturated sodium bicarbonate solution (60 mL) was added with stirring, followed by ethyl acetate (120 mL). The ethyl acetate layer was washed three times with water, dried with anhydrous sodium sulfate, and concentrated. A silica gel purification (CH2Cl2/CH 3 OH/NH 4OH = 8: 1: 0.1) of the mixture afforded Cbz-6-amino-hexyl-diamine-monoboc (0.19 g, 40%), which was directly allowed to react with boc-anhydride (1 equivalent) in methanol at room temperature for 2 h. The product 8 was purified on a silica gel column eluting with EtOAc/CH 2 C12 = 1: 2 to give 0.24 g (quantitative). 1H NMR (300 MHz, CDC13): 6 7.18 (s, 5 H), 5.11 (s, 2 H), 4.80 (br m, 2 H), 3.12 (m, 8 H), 1.40 (m, 24 H). DTB6diamine (9). The Cbz group of 8 (0.24 g, 0.49 mmol) was deprotected by adding 10% of Pd-C and hydrogen, using a hydrogen balloon under argon. The solution was stirred at room temperature for 30 min. After removing Pd-C by filtration, a quantitative yield of 6-amino-hexyl-diamine-diboc was obtained (0.17 g). The resulting 226 product was coupled with desthiobiotin by using NHS and DCC as described above for the preparation of 4, yielding DTB6diamine-diboc (0.25 g, 95%). Boc protecting groups were removed by 3 M HC1 in 6 mL of EtOAc/CH 3OH = 2: 1. Solvents were removed under reduced pressure and the resulting crude mixture was dissolved in 4 mL of water, basified with 2 M NaOH, and dried again. The product was extracted with 4 mL of methanol, yielding -180 mg of DTB6diamine (9), which still contained a small amount of NaCl. ESI-MS: m/e Calcd. (M + H+) 356.5, Found 356.3. H NMR (300 MHz, DMSO): 6 7.81 (s, 1 H, amide NH), 6.32 (s, 1 H, desthiobiotin NH), 6.14 (s, 1 H, desthiobiotin NH), 3.59 (m, 1 H), 3.48 (m, 1 H), 3.02 (q, 2 H), 2.81 (m, 4 H), 2.60 (t, 2 H), :2.03 (t, 2 H), 1.6 (m, 16 H), 0.98 (d, 3 H). DTB6Pt (10). To a solution of 9 (70 mg, 0.2 mmol) in water (2 mL) was added K2[PtCl 4 ] ( 84 mg, 0.23 mmol) in water (1 mL). The reaction mixture was stirred overnight at room temperature. The yellow precipitate was collected, washed with cold water, and dried to yield DTB6Pt (38 mg, 30%). ESI-MS: m/e Calcd. (M + H +) 622.2, Found 622.3. H NMR (300 MHz, DMF): 6 7.75 (t, 1 H, amide NH), 6.21 (s, 1 H, desthiobiotin NH),6.09 (br s, 1 H, Pt-NH), 6.04 (s, 1 H, desthiobiotin NH), 5.40 (br s, 2 H, Pt-NH2), 3.74 (m, 1 H), 3.62 (m, 1 H), 3.18 (m, 2 H), 3.02 (m, 1 H), 2.61 (m, 3 H), 2.20 (t, 2 H1-),1.91 (br m, 1 H), 1.6 (m, 16 H), 1.02 (d, 3 H). 1, 4-Diazidobutane (11) (Scheme 6.3). Sodium azide (NaN 3 ) (4.3 g, 66 mmol) was added to a solution of 1,4-dibromobutane in 40 mL of DMF and the mixture was stirred at 80 °C for 20 h. The solution was cooled to room temperature and water (20 mL) was added to solublize NaN 3 and NaBr. The product was extracted twice with diethyl ether (Et2O) (20 mL) and the combined ether layer was washed twice with 20 mL water. The resulting ether layer was dried with anhydrous sodium sulfate and concentrated under 227 reduced pressure to give 2.8 g (80%) of 11 as colorless oil. H NMR (300 MHz, CDC13): 3.27 (m, 4 H), 1.71 (m, 4 H). 1, 4-Azidoaminobutane(12). Triphenylphosphine (5.1 g, 19 mmol) was added in small portions to a solution of 11 (2.8 g, 20 mmol) in 30 mL of Et2 O/EtOAc = 1: 1 and 24 mL of 5% HC1 on an ice bath for over 1 h. Following an additional 24 h incubation at room temperature, the aqueous layer was separated and washed twice with dichloromethane (15 mL). The pH of the solution was adjusted to -12 with NaOH and the product was extracted three times with dichloromethane (15 mL). The resulting solution was dried with anhydrous sodium sulfate and concentrated under slightly reduced pressure. Finally, azidoaminobutane was further purified by distillation at - 60 °C under reduced pressure to yield 0.8 g (35%). 'H NMR (300 MHz, CDC13):6 3.25 (t, 2 H), 2.71 (t, 2 H), 1.60 (m, 2 H), 1.51 (m, 2 H), 1.25 (s, 2H). Pt4N3 (13). K[PtC13(NH3)] was kindly provided by Dr. Song in our lab. K[PtCl3 (NH3 )] (184 mg, 0.50 mmol) in water (2.5 mL) was added to a solution of 12 (57 mg, 0.50 mmol) in ethanol (2 mL). The reaction mixture was stirred overnight at room temperature. The dark yellow precipitate was collected and dissolved in 2 mL of DMF. Insoluble black solid was removed by centrifugation and 10 mL of water was added to the DMF solution to obtain yellow Pt compound 13 (20 mg, 15%). 1H NMR (300 MHz, DMF): 6 4.92 (br s, 2 H, Pt-NH 2), 4.22 (br s, 3 H, Pt-NH 3 ), 3.39 (t, 2 H), 2.80 (m, 2 H), 1.82 (m, 2 H), 1.65 (m, 2 H). DNA Blot and Desthiobiotin Detection. Indicated amount of normal DNA or damaged DNA was blotted onto a highly positive charged membrane, Biodyne B (Pall Corp.), with a vacuum manifold. After air-drying for at least 30 min, the membrane was blocked with a blocking solution (0.5% casein and 0.5% SDS in PBS) for 5 min with 228 constant shaking and incubated with the same solution containing 10 g of alkaline phosphatase (AP)-conjugated streptavidin for 20 min. Following the incubation, the blot was washed once with the blocking buffer, three times with a washing buffer (0.5% SDS in PBS), and twice with an assay buffer (0.1 M diethanolamine, pH 10, 0.1 mM EDTA). Chemifluorescence-based detection was performed according to the manufacturer's protocol with ECF (Amersharm) as an AP substrate. Cytotoxicity Assay. Cell lines used in this study were all obtained from ATCC; HeLa, HeLa S3, and Ntera II. For evaluation of cytotoxicity of prepared platinum compounds, 500-1000 cells were seeded in each well of a 96-well plate in 0.1 mL of appropriate medium. On the following day, platinum compounds were added to cells at concentrations between 0.1 to 200 M. Stock solutions were freshly prepared each time with indicated solvent. The cells were incubated with the compounds for 3 days before cell viability was determined by the MTT (3-(4',5'-dimethylthiazol-2'-yl)-2,5diphenyltetrazolium bromide) colorimetric assay. A 20 L portion of MTT solution in PBS (5 mg/mL) was added to each well containing treated cells and incubated for 4 h. The medium was removed and crystals formed were dissolved in 0.2 mL of DMSO. The optical density (OD) at 550 nm was measured by using a plate reader. Results and discussion Synthesis of Desthiobiotin-ConjugatedPlatinum Agents. Attempted synthesis of a biotin-conjugated cisplatin analog was problematic due to the sulfur atom of the biotin moiety, which forms a strong covalent bond with platinum. Desthiobiotin (DTB) is a biotin derivative lacking the sulfur atom. Although desthiobiotin binds less tightly to biotin-binding proteins such as avidin and streptavidin (3), it still possesses a strong binding affinity to streptavidin (Kd = ~1 pM) and is used in many biological 229 applications. Therefore, desthiobiotin is an ideal hapten molecule for the conjugation to platinum compounds. An initial attempt to synthesize DTB-Pt compound used desthiobiotin-amido-hexylamine (2), which is allowed to react directly with [PtC13(NH3)]* in order to make the cis form of Pt[C12(NH3)(2)]. Although a small amount of expected product was identified by a mass analysis, we were unable to purify it from a significant quantity of unidentified side products. An ethylenediamine moiety has been successfully used as a platinum chelating ligand in numerous syntheses (4,5). Desthiobiotin was then conjugated to platinum with two different strategies. First, ethylenediamine was added to desthiobiotin by a simple amide bond formation, followed by an amine deprotection as illustrated in Scheme 6.1. The chelating reaction of compound 4 to K2[PtCl4 ] afforded fairly pure platinum complex 5 without complicated purification steps. To eliminate the possibility of peptide bond adjacent to the platinum center interacting with the metal, DTB6diamine 9 was synthesized from 6amino-hexanol as shown in Scheme 6.2. Overall, the reaction of ethylenediamine (en) with [PtCl4]2*toform [PtCl2(en)] gives much better yield and easier purification of products than those of monoamine ligand reactions with [PtC13(NH3)] + to make [PtC12(NH3 )(NH2R)]. Previously, however, several monoamine ligands have been conjugated to [PtCl3(NH3)] + successfully (6,7). The solubility of the ligand might contribute to the outcome of the reaction, as desthiobiotion is highly soluble in aqueous solutions. Characterizationof Desthiobiotin-ConjugatedPlatinum Agents. DTB9Pt was allowed to react with a short oligonucleotide to test its ability to attack and form platinum adducts with DNA. 18TGGT (5'-CCTCTCCTGGTCTCTTCC-3'), where only one platinum GG reaction site exists, was incubated with DTB9Pt. 18TGGT-DTB9Pt containing a 1,2 d(GpG) intrastrand cross link was purified by ion-exchange HPLC. The 230 HPLC elution profile of the reaction mixture of DTB9Pt with 18TGGT was similar to that of cisplatin (data not shown). The platinated oligonucleotide was verified by mass analysis; ESI-MS: m/e Calcd. (M) 5932.8, Found 5933.3. Radioactively labeled 18TGGT- DTB9Pt was incubated with streptavidin-coated magnetic beads. The beads remained radioactive even after extensive washes with solutions containing a high concentration of NaCl, indicating strong desthiobiotin-strepavidin and DTB9Pt-DNA interactions. Preliminary studies indicate that DTB9Pt-modified DNA specifically interacts with HMGB1 protein and is also able to inhibit in vitro transcription (data not shown). These data suggest that DTB9Pt is comparable with cisplatin with regard to in vitro activity. After sheared salmon sperm DNA was treated with DTB9Pt, the platinum content of DNA was measured by atomic absorption spectroscopy (AAS). platinated IDNA samples using various DTB9Pt levels were blotted onto the Biodyne membrane and the desthiobiotin level was determined by AP-streptavidin as described in Experimental Procedures. Chemifluorescence signals were shown in DNA platinated with DTB9Pt, but not cisplatin (Figure 6.1A). 0.5 fmol of platinum adducts is readily detected with good linearity. Samples were also prepared with DTB6Pt, followed by DNA blot analysis as shown in Figure 6.1B. Detection of DTB6Pt adduct was approximately ten times less sensitive compared to that of DTB9Pt adduct. The short linker of DTB6Pt must be responsible for the weak interaction of desthiobiotin of DTB6Pt with streptavidin. Chemiluminescence-based detection was also applied with different AP substrate (ECL, Amersharm) with similar sensitivity of DTB detection (data not shown). A duplex DNA containing a biotin moiety at the 5' end was also used for the AP-streptavidin detection (data not shown). Although the detection limit of biotin-DNA (< 0.1 fmol) was similar to that of desthiobiotin-DNA, biotin detection afforded a better linearity than desthiobiotin. 231 Cytotoxic activities of DTB9Pt and DTB6Pt were examined with a MTT cell proliferation assay. IC50 values of both compounds were compared with those of cisplatin in various cell lines (Table 6.1). The complexes are over 100 times less cytotoxic compared to cisplatin. Despite the fact that desthiobiotin-Pt compounds effectively mimic biologically active platinum drugs in vitro, the inability to act on an intact cell restricts their values as model compounds to study platinum based anti-cancer agents. The high reactivity of the ethylenediamine-chelated platinum compound might explain low toxicity, where activated DTB-Pt complexes react with other cellular components prior to reaching DNA. DTB9Pt(IV)compound, which has to be processed into Pt(II) in a cell to act as a toxic reagent, was also prepared with an aim to delay the activation of DTB-Pt complexes (data not shown). The Pt(IV) compound, however, did not show much improved biological activity compared to the corresponding Pt(II) compound. Synthesis of Azide-ConjugatedPlatinumAgent. An azide moiety has been used in a number of systems owing to its bioorthogonal reactivity. It does not exist in nature and is unreactive with biologically relevant functional groups. The unique reactivity of the azide to phosphines and alkynes allows it as a chemical reporter molecule for bioconjugation (8,9). An azide-conjugated platinum compound was synthesized as shown in Scheme 6.3. Azidoaminobutane 12 was obtained from dibromobutane as previously described (10). The synthesis of PtC12(NH3)(NH2(CH2) 4N 3) was achieved by reacting azidoaminobutane 12 with [PtC13(NH3)]*. Unlike desthiobiotin, the azide is not soluble in aqueous solution, which might contribute to the successful reaction as discussed above. A preliminary study with Pt4N3 13 indicated that the compound is only 6 times less toxic than cisplatin against HeLa S3 cells. 232 Conclusion [Pt(en)C1 2] analogs containing a desthiobiotin moiety with different linkers (DTB9Ptand DTB6Pt) were successfully synthesized. Although the compounds display in vitro activities similar to those of cisplatin, their possible applications in live cells are limited due to poor cytotoxicity. PtC12(NH3)(NH2(CH2)4N3) (Pt4N3, 13), however, shows better biological activities than DTBPts. Future work includes a search for a better conjugation method or alternative hapten molecule. Acknowledgments :[ thank Professor C. X. Zhang and Dr. K. R. Barnes for experimental guidance and helpful discussions. 233 References 1. Molenaar, C., Teuben, J. M., Heetebrij, R. J., Tanke, H. J., and Reedijk, J. (2000) J. Biol. Inorg. Chem. 5 2. Kalayda, G. V., Zhang, G., Abraham, T., Tanke, H. J., and Reedijk, J. (2005) J. Med. Chem. 48, 5191-5202 3. Hirsch, J. D., Eslamizar, L., Filanoski, B. J., Malekzadeh, N., Hougland, R. P., Beechem, J. M., and Hougland, R. P. (2002) Anal. Biochem. 308, 343-357 4. Robillard, M. S., Valentijn, A. P. M., Meeuwenoord, N. J., van der Marel, G. A., van Boom, J. H., and Reedijk, J. (2000) Angew. Chem. Int. Ed. 39, 3096-3099 5. Robillard, M. S., Bacac, M., van den Elst, H., Flamigni, A., van der Marel, G. A., van Boom, J. H., and Reedijk, J. (2003) J. Comb. Chem. 5, 821-825 6. Zhang, C. X., Chang, P. V., and Lippard, S. J. (2004) J. Am. Chem. Soc. 126, 6536- 6537 7. Kane, S. A., and Lippard, S. J. (1996) Biochemistry 35, 2180-2188 8. Kohn, M., and Breinbauer, R. (2004) Angew. Chem. Int. Ed. 43, 3106-3116 9. Speers, A. E., Adam, G. C., and Cravatt, B. F. (2003) J. Am. Chem. Soc. 125, 4686- 4687 10. Lee, J. W., Jun, S. I., and Kim, K. (2001) Tetrahedron Lett. 42, 2709-2711 234 Table 6.1. IC50values (M) for cisplatin, DTB9Pt, and DTB6Pt Cell lines HeLa HeLa S3 GM fibroblast 1.1 0.35 2.0 DTB9Pt >100 70 >100 DTB6Pt >100 60 Cisplatin Standard deviation is -20%. Data were collected from more than two independent experiments. 235 0 HN /NH 0 OH /Boc +H2N H 0 HN /NH H .N H i Boc N, H 1 0 HN /KNH Boc 0 H /Boc c,d, HONN + H 02 3 0 HN NH N. NH)4N .H 0 HN 0 - 4 Cl "NH ;,N.° Cl Pt NH H~~H 0N-N ""Pt"' 0 5 Scheme 6.1. Synthesis of DTB9Pt (5). Reagents and conditions: (a) HATU, NMM, DMF; (b) conc. HC1, EtOAc; (c) DCC, NHS, TEA, CH 3OH; (d) conc. HC1, EtOAc; (e) K2 [PtC14], H 2 0. 236 + a0 H2N OH CIa o H H -OH CbzN'N b > c, Cbz/No 7 6 CbzN~ z\ CbzN /N, _// H H 8 Boc e,f,g Boc 0 HN / NH \> ,N N-NH2 h, H 09 HN NH 10 Scheme 6.2. Synthesis of DTB6Pt (10). Reagents and conditions: (a) TEA, CH 3OH; (b) TPAP, MMO, CH2C12; (c) boc-ethylenediamine, Na(CN)BH 3, CH 3OH/AcOH; (d) bochydride, CH 3OH; (e) 10% Pd-C, H2; (f) biotin, DCC, NHS, TEA, CH 3OH; (g) conc. HC1, EtOAc/ CH 3OH; (h) K2[PtC14 ], H 2 0. 237 B'---Br a N3 1 N3 b N3" 11 C NH2 12 ,Pt.NN3 ClI\ ,/NH3 H2 13 Scheme 6.3. Synthesis of Pt4N 3 (13). Reagents and conditions: (a) NaN 3, DMF (H 20), 80 °C, 20 h; (b) Ph 3 P, Et 2 O/EtOAc/5%HC1; (c) K[PtC1 3 (NH 3)], H 2 0/EtOH. 238 A I II -C I I_ Il _ C_··_ ---- II·- __- DNA damaged by Cisplatin DNA damaged by DTB9Pt - ---_ -LC~~~ 100 50 20 - - -- -- --- -- -- ---- -- ·li- --- - 10 5 2 1 0.5 I [Pt] (fmol) B @ DNA damaged by DTB6Pt 0 DNA damaged by DTB9Pt 20 10 5 2 1 0.5 0.2 0.1 [Pt] (fmol) Figure 6.1. DNA blot analysis. DNA damaged by indicated platinum compound was blotted on to a positive charged membrane. Quantities of platinum adducts on each blot were indicated in an fmol scale. 239 Biographical Note The author was born Yongwon Jung on February 23, 1977, to Nojin Jung and Kwiney Hwang, in Pyeongtak, Korea, where he grew up with four sisters. He attended Kyungki Science High School in 1992 away from his home town and his interest in chemistry started to grow during these two years of fun time. The author then moved to Korea Advanced Institute of Science and Technology (KAIST) in Daejon ,Korea, where he received a 1998B.Sc.degree in Chemistry and a 2000M.S. degree in Biochemistry under the direction of Professor Younghoon Lee. During his Master, he studied the properties of various electron transfer proteins on a modified gold electrode. In September 2000, the author began graduate studies in biological chemistry at MIT. He joined the lab of Professor Stephen J. Lippard where he studied the molecular mechanism of platinumbased anticancer drugs. While at MIT, he also mentored the laboratory studies of two undergraduate students, Sarah Simmons and Cindy Yuan. Following graduation, he plans to join KRIBBback in Korea as a research scientist. 240 Yongwon Jung Education * Massachusetts Institute of Technology, Cambridge, MA. Ph.D. in Biological Chemistry, 2005. Thesis advisor: Prof. Stephen J. Lippard * Korea Advanced Institute of Science and Technology, Taejon, Korea. M.S. in Biochemistry, 2000 Thesis advisor: Prof. Younghoon Lee And B.S. in Chemistry, 1998. Publications 1. Jung, Y. and Lippard S.J., "RNA polymerase II blockage by platinum DNA damage: polyubiquitylation of stalled polymerase", submitted. 2. Jung, Y. and Lippard S.J., "Multiple states of stalled T7 RNA polymerase at DNA lesions generated by platinum anticancer agents", J. Biol. Chem. (2003),278, 52084-52092. 3. Jung, Y. and Lippard, S.J., "Nature of full-length HMGB1 binding to cisplatinmodified DNA", Biochemistry (2003), 42, 2664-2671. 4. Park, J.W., Jung, Y., Lee, S. J., Jin, D.J., and Lee, Y., "Alteration of stringent response of the Escherichia coli rnpB promoter by mutations in the -35 region", Biochem. Biophys. Res. Commun. (2002), 290, 1183-1187. 5. Jung, Y., Mikata, Y., and Lippard, S.J., "Kinetic studies of TATA binding protein interaction with cisplatin-modified DNA", J. Biol. Chem. (2001), 276, 43589-43596. 6. Jung, Y., Kwak, J., and Lee, Y., "High-level production of heme-containing holoproteins in Escherichia coli.", Appl. Microbiol. Biotechnol. (2001), 55, 187-191. Presentations Jung, Y. and Lippard S.J., "Multiple states of stalled T7 RNA polymerase at DNA lesions generated by platinum anticancer agents" 9 th International Symposium on Platinum Coordination Compounds in Cancer Chemotherapy, New York, October 2003.

Cellular Responses against DNA Damaged by Platinum Anticancer Drugs by Yongwon Jung

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