MISMATCH REPAIR AND MAINTAINING GENOME STABILITY

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The goal of this project is to understand the role of mismatch repair in maintaining genome stability in S. cerevisiae. In organisms ranging from E. coli to humans, mismatch repair has been shown to play an important role in preventing spontaneous point mutations as well as insertion and deletion mutations in repetitive DNA sequence. In humans, defects in mismatch repair have been direct correlated to hereditary non-polyposis colorectal cancer, a disease that is thought to affects as much as 0.5% of the population. Additional studies in E. coli and S. cerevisiae suggest that mismatch repair plays an important role in preventing chromosomal rearrangements by rejecting recombination intermediates that contain base pair mismatches. In E. coli, an in vitro mismatch repair system has been reconstructed using purified components. While this system has provided much information regarding repair mechanisms, it has not addressed how DNA substrates are recognized at the molecular level or how mismatch repair proteins act to modulate genetic recombination events. These issues will be addressed by undertaking a detailed genetic and biochemical analysis of S. cerevisiae MSH2 (mutS homolog #2). The MSH2 gene is highly homologous to mutS, a critical component of the E. coli mutHLS mismatch repair system that binds to base pair mismatches in vitro. Initial studies have shown that Msh2 is the major mismatch recognition protein in S. cerevisiae, is involved in preventing spontaneous point mutations, insertion and deletion mutations in highly repetitive DNA sequences, and modulates gene conversion tracts. Mutational analysis will be performed on the MSH2 gene to identify dominant and conditional mutants. These mutants will be examined for their effect on the frequency of a number of events, including spontaneous mutations, deletions and insertions at repetitive DNA sequences, mitotic and meiotic recombination at a number of homologous and ectopic sites, and chromosomal rearrangements. Mutants will be analyzed biochemically for binding to DNA substrates containing mismatches and for interaction with other mismatch repair components. Dominant and conditional mutants will then be used in genetic suppressor analysis. This analysis will be performed in both mitotic and meiotic assays, with the hope that specific suppressor will be identified. Synthetic lethality screens using specific msh2 mutations will be initiated, as well as a synthetic lethality screen aimed at identifying mutations that are lethal in recombination deficient, mismatch repair proficient strains, such mutants could be involved in marking DNA for strand specific repair. Studies performed in both S. cerevisiae and E. coli have suggested that mismatch repair in involved in preventing chromosomal rearrangements. This hypothesis will be tested in S. cerevisiae using both novel and established assays. These assays will be used to determine whether msh2 and other mismatch repair mutants affect the frequency of gene conversion and crossing over in both mitosis and meiosis for homologous and homologous sequences placed at either homologous or ectopic positions. In addition the effect of these mutations on a novel illegitimate recombination assay will be tested.