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) suppresses mutations that result from mispaired bases in DNA and limits recombination between related DNA sequences containing base differences reducing the frequency of aberrant recombination events. Thus, MMR defects increase 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 non-polyposis colon cancer is due to inherited defects in MMR and many sporadic cancers appear MMR defective, yet the genetic consequences of MMR defects are not fully understood; And, 2) Many chemotherapy agents damage DNA and MMR defects can result in resistance to some of these agents so understanding MMR could lead to improvements in the efficacy of these agents as well as ways to circumvent MMR defect-mediated resistance. 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 provide insights into the genetics of human cancer susceptibility. The following lines of experimentation will be carried out: 1) Genetic studies will identify MMR genes, MMR proteins that interact and provide mutations for use is dissecting the biochemical properties of MMR proteins; 2) Biochemical studies of individual MMR proteins including the MSH2-MSH3, MSH2-MSH6, MLH1-PMS 1 and MLH 1-MLH3 complexes, RPA, PCNA, DNA polymerase 8, RFC, EXO1, RAD27 and 2 new exonucleases will be continued to determine the roles these proteins play in MMR; 3) Higher order protein complexes that function in MMR will be identified and studied; And, 4) partial and complete MMR reactions will be reconstituted in vitro using purified proteins. 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 genetic and biochemical insights that can be applied to the study of the genetics of human cancer susceptibility.
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