Effect of Cancer-Associated Mutations on MLH1 Interaction with Exonuclease Gautam Mankaney
advertisement
Effect of Cancer-Associated Mutations on MLH1 Interaction with Exonuclease Gautam Mankaney Mentor: Dr. Andrew Buermeyer Howard Hughes Medical Internship CANCER Cancer • Uncontrolled proliferation of cells • 2nd leading cause of death (500 thousand/ yr) New Cancer Incidents by State Colorectal Cancer • 3rd most common cancer (10.7%) • $8.4 billion in treatment costs Causes • Cellular mutations • Inherited or Sporadic • 3-5% cases linked to Lynch Syndrome (HNPCC) Lynch Syndrome (HNPCC) •80% develop colorectal cancer other cancers include: kidney, stomach, ovary, small bowel, pancreas , & bile duct •Mismatch Repair Deficiency Detected MMR Genes in Lynch Syndrome Families 100 Detected Mutations (%) 90 80 70 60 50 40 30 20 10 0 MLH1/ MSH2 MSH6 MMR Genes PMS2 DNA Repair Mechanisms - Mismatch Repair (MMR) DNA Damage •Environmental (carcinogens, UV light) •Metabolic activities (free radicals, replication) DNA Mismatch Repair •Evolutionarily conserved process •Fidelity of DNA replication a) Base substitution, insertion, and deletion mismatches and loops b) DNA lesions - environmental and intracellular stress c) Apoptosis •MMR loss - multistage carcinogenesis PROKARYOTIC MMR mismatch 5’ 3’ 3’ 5’ CH3 5’ nick CH3 MutS, MutL, MutH 3’ nick mismatch mismatch 5’ 3’ 3’ 5’ CH3 5’ 3’ 3’ 5’ CH3 CH3 Exonuclease - ExoVII or RecJ HelicaseII 5’ 3’ 3’ 5’ CH3 CH3 Exonuclease - ExoI HelicaseII 5’ 3’ 3’ 5’ CH3 CH3 DNA Polymerase III DNA Ligase DNA Polymerase III DNA Ligase 5’ 3’ 3’ 5’ CH3 CH3 CH3 MutLα – Understanding the Structure •MLH1 – PMS2 ExoI MLH1 - functional domains ATP-binding/ hydrolysis Dime r interface ss DNA binding 3 241 492 621 711 756 Linker PMS2, EX O1 inte raction C-terminal homology Research Question Compared to MLH1 wildtype and and non-pathogenic polymorphisms, how well does ExoI interact with certain MLH1 mutants? - L582V, K751R, L607H, and R755W -putatively associated with Lynch Syndrome -do not affect MLH1 protein stability -do not affect MLH1 - PMS2 interaction MLH1 - functional domains Hypothesis Compared to MLH1 wildtype and MLH1 non-pathogenic polymorphisms, L582V, K751R, ATP-binding/ hydrolysis Dime r interface ss DNA binding 3 621 L607H, and R755W241will show a decreased492ability in binding EXO1 711 756 Linker PMS2, EX O1 inte raction - in vitro assays that measure interaction capabilities C-terminal homology Approach 1. Construct in vitro expression vectors containing coding regions to be expressed - MLH1 Wildtype - MLH1 Mutants: L582V, L607H, K751R, R755W - ExoI 2. Find a way to probe for ExoI -express protein and test antibody 3. Perform in vitro co-immunoprecipitation assays with ExoI, PMS2, and MLH1 variants Plasmid Construction hMLH1 wt pCMV •Excision of cDNA by restriction digestion hMLH1 hMLH1 wt mutant •Gel isolation of cDNA hMLH1 mutant pCMV •Ligation into linear pCite vector •Restriction digests (screening) and sequencing hMLH1 hMLH1 mutant pCite Constructing the Plasmid Isolate Fragments (Three Way Ligation) hMLH1 hMLH1 wt MLH1(part) hMLH1 hMLH1 mutant MLH1(part containing mutation) mutant pCite Plasmid Screening hMLH1 hMLH1 hMLH1 hMLH1 2.4 kb mutant mutant restriction enzyme (Xho1) pCite 3.8 kb Restriction Digests ladder 1 10,000 kb 2 3* 4 5 6* 7 6000 kb 5000 kb 4000 kb 3000 kb 2500 kb 2000 kb ladder 15 10,000 kb 6000 kb 5000 kb 4000 kb 3000 kb 2500 kb 2000 kb 16 17* 18* 19 20 QuickTime™ and a TIFF (Uncompressed) decompresso are needed to see this picture. K751W QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. L582V 21* L607H 8 9 10 R755W 22* 23* 24 11 12 13 14* Co-immunoprecipitaton hMLH1 pCite PMS2 hEXO1 pCITE pCITE Antibody – PMS2 Antibody Binding Beads Transcription Translation Western Blot Wash Coimmunoprecipitation Example Co-immunoprecipitation QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. •Qualitative Measurement Exonuclease Detection hEXO1 pCITE Questions 1. Is protein being produced? protein Transcription 2. Is there a problem with the detection method? Exonuclease ≈ 105 Translation MCF-7 30ul 15ul 10ul 5ul kD MCF-7 30ul 15ul 10ul 5ul 250 150 100 75 U.S Biological NeoMarkers (mouse monoclonal Ab) (mouse monoclonal Ab) 50 Flagging hEXO1 •Octapeptide - DYKDDDDK •Polymerase Chain Reaction (PCR) hEXO1 hEXO1 Flag-Exonuclease Detection 30ul 15ul 10ul 5ul Flag-Exonuclease 30ul 15ul 10ul Exo Exo 4.5ng 2.2ng 1.5ng .75ng 1.5ng 4.5ng 2.2ng 1.5ng 1.5ng 250 Anti-Flag 150 15mg protein 100 75 rabbit polyclonal anti-Flag 50 Dr. Binghui Shen City of Hope National Medical Center and Beckman Research Institute Summary Constructed prokaryotic transcription vectors For MLH1 WT, MLH1 mutants, ExoI Insufficient antibody sensitivity to ExoI with two different mouse monoclonal Ab’s Added flag peptide to amino terminal of ExoI reading frame Insufficient antibody sensitivity to ExoI with Anti-Flag Future Studies 1. FLAG carboxyl end of hEXO1 2. Try a different epitope tag 3. 35S labeled Methionine 4. 1Glutathione-S-transferase protein-protein interaction assay -GST fusion protein (PMS2) -35S labeled ExoI 5. 2Two Hybrid Assay - PMS2 fused to a binding domain - ExoI fused to activation domain 1Schmutte, C., M. M. Sadoff, S. Guerrette, S. Acharya, and R. Fishel. Interactions of the human exonuclease I with DNA mismatch repair proteins hMSH2, hMSH3 and hMLH1. J. Biol. Chem., in press. 2 Tran, P. T., J. A. Simon, and R. M. Liskay. Interaction of EXO1 with components of MutLα in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA, in press. Thank You! •The Buermeyer Lab QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Dr. Andrew Buermeyer Dr. Scott Nelson •Howard Hughes Medical Institute •Undergraduate Research, Innovation, Scholarship & Creativity (URISC) •Dr. Kevin Ahern CTD NTD CTD NTD DNA Mismatch Repair (MMR) 2) The PMS2 mutants will show a decreased ability to bind MLH1 compared to PMS wildtype Perform Ligations Using T4 DNA Ligase Schmutte, C., M. M. Sadoff, S. Guerrette, S. Acharya, and R. Fishel. Interactions of the human exonuclease I with DNA mismatch repair proteins hMSH2, hMSH3 and hMLH1. J. Biol. Chem., in press. 74a. Tran, P. T., J. A. Simon, and R. M. Liskay. Interaction of EXO1 with components of MutLα in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA, in press.
