Functional consequences of NLS mutations in human MLH1 Alex Dukes Dr. Andrew Buermeyer Department of Environmental & Molecular Toxicology Oregon State University HHMI Program, Summer 2007 The Importance of Mismatch Repair (MMR) Cellular mechanism for preventing DNA mutations, resulting from: Replication errors Recombination pathways Exogenous DNA damaging agents Necessary to trigger apoptosis in event of extensive DNA damage MMR and Colon Cancer Colorectal cancers account for 10% of all new cancers Lynch Syndrome, a type of hereditary colon cancer (HNPCC) Caused by genetic defects in MMR genes ©2005 American Cancer Society, National Cancer Institute A Component of MMR: MutLα Heterodimer composed of MLH1 and PMS2 proteins In the absence of MMR, cells display: Increased mutation rate Decreased apoptotic response to genotoxins Mutations in MLH1 account for 30-40% of Lynch Syndrome cases Two Mutations of Interest Missense mutations both identified in cancer patients Nuclear localization sequence (NLS): Acts as a cellular “password” to allow protein into nucleus via the nuclear pore amino acids 470 – 474 in MLH1 Image courtesy of: Wu, X., et al, Dimerization of MLH1 and PMS2 Limits Nuclear Localization of MutL. Molecular and Cellular Biology, 2003. 23(9): p. 3320–3328 Image courtesy of : www.scripps.edu/.../proj/NPC/NucleusWhite.gif Mutation #1 R474Q Amino acid 474 Replaces arginine (R) with glutamine (Q) Charged Charged Images courtesy of: www.contexo.info/.../images/aminoacidsweb.gif Mutation #2 R472I Amino acid 472 Replaces arginine (R) with isoleucine (I) Charged Non-polar (more deleterious?) Images courtesy of: www.contexo.info/.../images/aminoacidsweb.gif Project Objectives Examine two NLS mutations in MLH1 and identify the phenotypic consequences of each Experimental Methods Transient transfection Stable transfection The Transient Transfection Is the mutant MutLα heterodimer stable? Does the mutation interfere with normal localization patterns? Plasmids encoding mutated MLH1 and PMS2 are introduced into MutLα- deficient cells Cells assessed to determine protein expression and localization pattern Nuclear MutLα present MutLα absent Cytoplasmic The Transient Transfection Lipid-based transfection The Stable Transfection Does the mutation result in reduced MMR function? Plasmid encoding mutated MLH1 is introduced into MLH1- deficient cells Cells exhibiting reduced MMR function identified by: Increased mutation levels Loss of apoptotic response This assay also can be used to confirm the heterodimer stability and localization pattern found in the transient transfection. The Stable Transfection Electroporation Selection with cytotoxic G418 Positive Control Negative Control R472I R474Q Plating from the Stable Transfection Ouabain Assay 6-TG Assay Mutation frequency Survival following DNA damage response Pass 1:10 Pass 1:10 Freeze cell lines Interpretation of possible outcomes Hypothesis: Both mutations interfere with localization and cause MMR deficiency. Localization MMR Phenotype Interpretation of Results Cytoplasmic Deficient Mutation interferes with localization causing MMR deficiency Cytoplasmic Proficient Low levels of MutLα enter nucleus and are sufficient for MMR phenotype Nuclear Deficient Mutation interferes with enzymatic function or other protein activity, not localization Nuclear Proficient Mutation does not interfere with normal localization Transient Transfection Results 1 2 3 4 MSH6 PMS2 MLH1 Both R474Q and R472I mutants stabilize PMS2 Stable Transfection Results: Protein Expression Levels MLH1 accumulates similar to wild type MLH1 stabilizes PMS2 similar to wild type Stable Transfection Results: Cellular Localization Mutation #1: R474Q Mutation #2: R472I Both mutants exhibit nuclear localization Stable Transfection Results: Mutation Frequency MMR Deficient (HIGH mutant frequency) MMR Proficient (LOW mutant frequency) Stable Transfection Results: Cytotoxic Response MMR Deficient (HIGH survival) MMR Proficient (LOW survival) Summary of Results Localization? NUCLEAR Mismatch repair phenotype? PROFICIENT Possible Explanations 1 The mutation in MLH1 did not critically interfere with the NLS 2 A redundant NLS sequence allowed the protein into the nucleus (Another in MLH1? In PMS2?) Colon Cancer Implications Our functional assays show no reduced MMR activity Other lab groups obtained similar results for R474Q Clinical data is not conclusive in linking mutations to Lynch Syndrome CONCLUSIONS 1. The mutations are not pathogenic. 2. The mutations are moderately pathogenic and our assays are not sensitive enough. Acknowledgements Howard Hughes Medical Institute Dr. Andrew Buermeyer, mentor Dr. Kevin Ahern, program coordinator Generously funded by grants from the HHMI Program