Roles for Mismatch Repair Proteins in Maintaining Genome Stability

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Project Summary DNA mismatch repair (MMR) systems act to excise misincorporation errors that occur during DNA replication. In eukaryotes MSH proteins recognize these errors in the context of base-base and insertion/deletion mismatches and recruit MLH complexes to form ternary complexes that work with replication factors (RPA, RFC, PCNA) and Exo1 to excise the newly replicated DNA strand through the mismatch site. This is followed by DNA re-synthesis steps. MMR factors also recognize mismatches that form during strand invasion steps in homologous recombination; they recruit a helicase complex that unwinds (rejects) recombination intermediates and allows a new homology search. In addition, subsets of MMR factors act in meiosis to resolve recombination intermediates into crossovers (COs). In baker’s yeast the majority of meiotic COs are formed in an interference-dependent pathway in which double Holliday junctions (dHJs), thought to be stabilized by Msh4-Msh5, are resolved through the actions of STR helicase/topoisomerase, Exo1 nuclease, and the MutLγ (Mlh1-Mlh3) endonuclease. Our work is focused on developing molecular models to explain how the different MSH and MLH factors act in the above pathways. This work will enable us to understand how molecular defects in these factors underlie human infertility and hereditary forms of colon cancer, and how chromosomal rearrangements can lead to disease. We will test these ideas through three distinct research themes. In Project 1 we are studying how conformational changes in MLH proteins, mediated by ATP binding and hydrolysis, are linked to strand specificity steps in MMR and meiotic recombination. We will use genetic (mutations in intrinsically disordered domains in Mlh1-Pms1 and Mlh1-Mlh3 and force dimerization of MLH proteins), biochemical (in vitro reconstitution reactions to determine specific roles for MLH proteins in MMR and mass-spectrometry) and single-molecule (examine diffusion along DNA and how proteins bypass barriers) approaches. Project 2 is focused on understanding how MutLγ acts to resolve dHJs in the ZMM pathway. Our work in the current grant period is consistent with MutLγ endonuclease being activated in MMR and meiotic crossing over through the formation of a MutLγ filament. We will use this information and biochemical, mass spectrometry, and genetic methods that take advantage of our identification of mlh3 separation of function mutants to identify MutLγ interacting factors. Our early work encourages us to initially focus on MutLγ interactions with the Exo1 nuclease, after which we will test identified factors alone and in combination for their ability to interact with MutLγ to cleave model HJ and dHJ substrates. Project 3 is aimed at understanding how the decision is made to repair or reject recombination intermediates. We will analyze how mutations in histone chaperones and deacetylases, separately and in combination, affect anti-recombination, and will employ an inducible system to provide a temporal and physical measure of these effects. This work will also encourage us to pursue a genome-wide screen to identify new factors that regulate the repair/rejection decision.