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 MutL? (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 MutL? acts to resolve dHJs in the ZMM pathway. Our work in the current grant period is consistent with MutL? endonuclease being activated in MMR and meiotic crossing over through the formation of a MutL? 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 MutL? interacting factors. Our early work encourages us to initially focus on MutL? interactions with the Exo1 nuclease, after which we will test identified factors alone and in combination for their ability to interact with MutL? 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.
Distinct groups of eukaryotic DNA mismatch repair (MMR) family proteins facilitate crossing over between homologs to ensure proper Meiosis I chromosome segregation, and act to prevent recombination between divergent DNA sequences that can lead to chromosomal rearrangements associated with diseases such as cancer. In humans, failures in meiotic chromosome segregation result in aneuploidy syndromes (at least 10% of human pregnancies), which are thought to occur as the result of Meiosis I and II errors, defective or inappropriate recombination, premature homolog separation, and defects in sister chromatid cohesion. Importantly, mutations in MMR genes have been found in about half of hereditary non-polyposis colorectal cancer (2-7% of all colon cancers) patients, and are thought be linked to infertility, underscoring the importance of obtaining new mechanistic understandings and molecular tools to establish allele pathogenicity.
