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Determining Phenotypes of Pathogenic Mismatch Repair Mutants

Determining Phenotypes of Pathogenic Mismatch Repair Mutants. Brett Palama Lab of Andrew Buermeyer, Ph.D. Dept. of Environmental/Molecular Toxicology. Research Goals.

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Determining Phenotypes of Pathogenic Mismatch Repair Mutants

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  1. Determining Phenotypes of Pathogenic Mismatch Repair Mutants Brett Palama Lab of Andrew Buermeyer, Ph.D. Dept. of Environmental/Molecular Toxicology

  2. Research Goals • Determine functional phenotypes of cells containing certain mutations in a key DNA mismatch repair protein which lead to human cancer • Aid the development of relevant treatment and prevention methods

  3. DNA Mismatch Repair (MMR) • Highly conserved pathway which helps ensure preservation of genomic integrity • May also play role in recognition of excessive DNA damage and subsequent signaling of apoptosis • Well-defined in prokaryotes; still lacking mechanistic information in eukaryotes

  4. G G A T T T T Mechanism for Mismatch Repair Recognition ! Selection Excision Resynthesis

  5. MLH1 MLH3 MLH1 PMS2 MLH1 PMS1 Substitutions Short Loops Long Loops MSH2 MSH6 MSH2 MSH3 Eukaryotic MMR Proteins • MMR carried out by two MutS homologs and three MutL homologs, each with varying heteroduplex specificity • Also dependent upon several replicative proteins (e.g. PCNA); excision mechanism still unknown

  6. Accounts for 2-5% of annual colorectal cancer cases • Characterized by microsatellite instability (MSI) • 70% of cases involve mutations of mlh1 or msh2 gene • Greatly increases likelihood of developing other cancers (endometrial, ovarian, etc.) Loss of MMR in Human Cancer • Germline mutations in one copy of a MMR protein are evident in a large majority of patients with Lynch Syndrome (HNPCC) • Pathogenic germline mutations in both copies of a MMR protein result in severe early-onset leukemia or lymphoma (lifespan < 5 yrs) • Inactivation of MMR (evident from MSI) also apparent in 13% of sporadic colorectal cancers

  7. Pathogenic Mutations in hMLH1 Specific interest of study is the hMLH1 protein (the non-redundant member in human MutL complexes) Map of hMLH1 protein, tentative domains, and pathogenic mutations

  8. Questions to Address • Which cellular phenotypes (error correction and DNA damage surveillance) are present/deficient in each mutant? • Are there pathogenic mlh1 mutants in which only one MMR function is inactivated (“separation of function” mutant)? Mutator phenotype = higher mutation frequency = increased chance of mutation in tumor suppressor genes/oncogenes = cancer DNA damage tolerance = higher DNA damage threshold = increased likelihood of propagation of damaged cells = cancer • What is the biochemical mechanism for the deficiencies introduced by mutations in mlh1?

  9. L749ter and the CTH Domain • Mutation which inserts a premature stop codon in the hMLH1 sequence, deleting the final eight amino acids of the polypeptide • Previous data shows that L749ter has a mutator phenotype, high DNA damage tolerance, and inability to stabilize PMS2 • Two mutations identified in human cancer are in the CTH domain (K751R and R755W) • Will these two mutations show phenotypes similar to those of L749ter?

  10. Project Outline Site-directed mutagenesis of hMlh1 cDNA Subcloning into mammalian expression vector Transfection into mlh1- (and pms2- in transient transfections) mouse embryo fibroblasts Functional Assays

  11. hMLH1 hMLH1 Site-directed mutagenesis Sequencing of cDNA pBS SK- pBS SK- Removal of cDNA by restriction digestion; blunting of cDNA; gel isolation of cDNA Ligation into linear, blunted pCMVBamNeo vector; Transformation into E.Coli hMLH1 Large-scale plasmid preparation Sequencing of cDNA hMLH1 pCMV BamNeo Mutagenesis/Subcloning DNA for use in functional assays!

  12. Functional Assays After subcloning into pCMVBamNeo and transfection into MEF cells, assays are done to determine functional characteristics of mutant MLH1 protein Functions To Be Studied: Error Correction DNA Damage Recognition PMS2 Stabilization PMS2 Subcellular Localization PMS2 Binding

  13. PMS2 alone PMS2 wt MLH1 PMS2 L749t PMS2 K751R PMS2 R755W MSH6 PMS2 MLH1 PMS2 Stabilization Evidence suggests that MLH1 has a stabilizing effect on PMS2. Question: Which pathogenic mutants are incapable of stabilizing PMS2? Approach: Transiently transfect double-knockout cells with mutant hMLH1 and PMS2; use SDS-PAGE and Western Blot to determine protein levels.

  14. Concluding Questions • Why do the mutations K751R and R755W show stabilization of hPMS2 while L749ter does not? • Are the mutations at 751 and 755 pathogenic by some other means? Or are they neutral polymorphisms? • Will other mutations in the CTH behave similar to 751 and 755?

  15. Acknowledgements • Buermeyer Lab • Hays Lab • URISC • HHMI

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