There are 3 ways in which mismatched bases arise in DNA: 1) Misincorporation during DNA replication; 2) Production of regions of heteroduplex DNA during genetic recombination; And 3) chemical damage to DNA and DNA precursors. Mismatch repair (MMR) limits recombination between related DNA sequences containing base differences or between short repeated sequences thus reducing the frequency of aberrant recombination events. Failure to repair mispaired bases increases the spontaneous mutation rate and also gives rise to altered recombination events. Understanding the mechanism of MMR will impact human health for a number of reasons: 1) Hereditary nonpolyposis colon cancer is due to inherited defects in MMR and many sporadic cancers appear MMR defective yet not all of the genes that underlie these diseases are known; 2) Many chemotherapy agents damage DNA and understanding MMR could lead to improvements in the efficacy of these agents; And 3) purification of MMR proteins will provide new reagents for detecting base changes in DNA which will be useful for genetic studies. The goal of this proposal is to identify Saccharomyces cerevisiae MMR proteins and understand how they catalyze MMR. Associated goals are to understand how MMR interacts with genetic recombination, how MMR contributes to the fidelity of DNA replication and if defects in MMR are responsible for inherited diseases. The following lines of experimentation will be carried out: 1) Genetic studies will identify MMR genes and help understand how they function in MMR; 2) Two-hybrid interaction analysis will be continued to identify and study genes encoding proteins that interact with MSH2, MSH3 and MSH6; 3) Biochemical characterization of an in vitro MMR system will be continued to identify and purify enzymes required for MMR; 4) Biochemical studies of individual MMR proteins including the MSH2-MSH3, MSH2-MSH6, MLH1-PMS1 and MLH1-MLH3 complexes, RPA, PCNA and EXO1 will be continued to determine the roles these proteins play in MMR. The ultimate goal of these experiments is to reconstitute MMR with purified proteins and determine the mechanism of this reaction. It is also anticipated that these studies will provide tools for identifying additional MMR genes in higher eukaryotes for use in studying MMR and the genetics of human cancer susceptibility.
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