Roles for Mismatch Repair Proteins in Maintaining Genome Stability

Mismatch repair (MMR) factors, which act to remove DNA replication misincorporation errors, also function in genetic recombination and in adaptation to stress. The latter two roles are the current focus of my research efforts. Research Area 1 is centered on crossing over, a process critical in most eukaryotes for the accurate segregation of homologous chromosomes in meiosis to form gametes. Most crossovers in baker’s yeast meiosis result from the biased resolution of double Holliday Junction (dHJ) intermediates in steps involving Exo1 and the Mlh1-Mlh3 MMR endonuclease, a process conserved in higher eukaryotes. Little is known about how seemingly symmetric dHJs are resolved in a biased manner. We developed a model to explain biased dHJ resolution in which the Exo1 protein protects nicks in or near dHJs from being ligated; this protection promotes subsequent resolution by the Mlh1-Mlh3 endonuclease. We are testing it by developing a method in yeast to map meiotic DNA nicks genome-wide in wild-type, exo1, and other mutant backgrounds. The data will be analyzed in combination with Exo1 chromatin localization maps and established maps for meiotic chromatin marks and double-strand break sites to obtain a model for crossover resolution that will be refined through analyses of mutants displaying defects in early to late steps of meiotic recombination. Area 2 focuses on an analysis of baker’s yeast strains containing incompatible MLH1 and PMS1 MMR alleles. Our work supports an incompatibility model in which an elevation in mutation rate contributes to adaptation to stress conditions through the acquisition of beneficial mutations. However, long-term fitness costs associated with an elevated mutation rate must be eliminated by genetic suppression or buffered by mating. We will determine if signatures of adaptation to MMR incompatibility can be directly observed in yeast populations by first screening for mutations in PMS1 which partially or fully restore compatibility with mlh1 alleles containing mutations in different functional domains. We will then utilize information from the 1,010 yeast genomes project to create a model for how Mlh1 and Pms1 incompatibilities arise and are eliminated during adaptation to stress. Area 3 is focused on quality control mechanisms that act in genetic recombination. In the previous cycle we identified several chromatin factors which play roles in homologous recombination template choice and donor template stringency. We hypothesize that one of these factors, the SIR histone deacetylase complex, plays a role in stabilizing repair intermediates during slow to repair events such as break-induced DNA replication (BIR) by forming filaments adjacent to extending recombination intermediates (D-loop). We propose to track in yeast the localization of the SIR complex during BIR and correlate its cellular localization with repair intermediates as measured by chromatin immunoprecipitation and D-loop capture and extension assays. Our work provides an understanding for how defects in genome integrity underlie human infertility, hereditary forms of colon cancer, fungal pathogenesis, and diseases resulting from chromosomal rearrangements.