1. 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

2. 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

3. 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

5. A Component of MMR: MutL α  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

6. 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 : www.scripps.edu/.../proj/NPC/NucleusWhite.gif 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

7. Mutation #1  Amino acid 474  Replaces arginine (R) with glutamine (Q)  Charged  Charged R474Q Images courtesy of: www.contexo.info/.../images/aminoacidsweb.gif

8. Mutation #2  Amino acid 472  Replaces arginine (R) with isoleucine (I)  Charged  Non-polar (more deleterious?) R472I Images courtesy of: www.contexo.info/.../images/aminoacidsweb.gif

9. Project Objectives  Examine two NLS mutations in MLH1 and identify the phenotypic consequences of each Experimental Methods  Transient transfection  Stable transfection

10. The Transient Transfection  Plasmids encoding mutated MLH1 and PMS2 are introduced into MutL α - deficient cells  Cells assessed to determine protein expression and localization pattern Is the mutant MutL α heterodimer stable? Does the mutation interfere with normal localization patterns? NuclearCytoplasmic MutL α present MutL α absent

11. The Transient Transfection Lipid-based transfection

12. The Stable Transfection  Plasmid encoding mutated MLH1 is introduced into MLH1- deficient cells  Cells exhibiting reduced MMR function identified by:  Increased mutation levels  Loss of apoptotic response Does the mutation result in reduced MMR function? This assay also can be used to confirm the heterodimer stability and localization pattern found in the transient transfection.

13. The Stable Transfection Selection with cytotoxic G418 Positive ControlNegative ControlR472IR474Q Electroporation

14. Plating from the Stable Transfection Freeze cell lines Ouabain Assay Mutation frequencySurvival following DNA damage response 6-TG Assay Pass 1:10

15. Interpretation of possible outcomes Localization MMR Phenotype Interpretation of Results CytoplasmicDeficient Mutation interferes with localization causing MMR deficiency Hypothesis: Both mutations interfere with localization and cause MMR deficiency. CytoplasmicProficient Low levels of MutL α enter nucleus and are sufficient for MMR phenotype NuclearDeficient NuclearProficient Mutation interferes with enzymatic function or other protein activity, not localization Mutation does not interfere with normal localization

16. Transient Transfection Results PMS2 Only R474Q R472I 1 23 4 Wild Type MSH6 PMS2 MLH1 Both R474Q and R472I mutants stabilize PMS2

17. Stable Transfection Results: Protein Expression Levels MLH1 accumulates similar to wild type MLH1 stabilizes PMS2 similar to wild type

18. Stable Transfection Results: Cellular Localization Mutation #2: R472IMutation #1: R474Q Both mutants exhibit nuclear localization

19. Stable Transfection Results: Mutation Frequency MMR Proficient (LOW mutant frequency) MMR Deficient (HIGH mutant frequency)

20. Stable Transfection Results: Cytotoxic Response MMR Proficient (LOW survival) MMR Deficient (HIGH survival)

21. Summary of Results  Localization? NUCLEAR PROFICIENT  Mismatch repair phenotype? Possible Explanations A redundant NLS sequence allowed the protein into the nucleus (Another in MLH1? In PMS2?) 1 2 The mutation in MLH1 did not critically interfere with the NLS

22. 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.

23. Acknowledgements  Howard Hughes Medical Institute  Dr. Andrew Buermeyer, mentor  Dr. Kevin Ahern, program coordinator Generously funded by grants from the HHMI Program