There are 3 ways in which mismatched bases arise in DNA: 1) DNA damage gives rise to mismatched bases. 2) Misincorporation during DNA replication produces mispaired bases. 3) Genetic recombination produces regions of heteroduplex DNA containing mispaired bases. The failure to repair mispaired bases in DNA substantially increases the spontaneous mutation rate and also gives rise to altered recombination events. Recent evidence indicates mismatch repair acts to prevent, or limit, recombination between related DNA sequences containing extensive base differences or between short repeated DNA sequences. Thus mismatch repair appears to reduce aberrant recombination events. Understanding the mechanism of mismatch repair has potential impact on human health for a number of reasons. 1) There are inherited human diseases that could be caused by defects in mismatch repair. These include diseases associated with high spontaneous mutation rates, cancers, like colon cancer that are associated with destabilization of CA repeat sequences and diseases in which mutations accumulate in mitochondrial DNA resulting in premature aging related syndromes. 2) Many chemotherapy agents act by damaging DNA and understanding how mismatch repair functions could lead to development of new ways of sensitizing cells to DNA damaging agents and a greater understanding of how cells become resistant to DNA damaging agents. 3) Purification of mismatch repair proteins will provide new reagents for studying DNA structure and detecting base changes in DNA which will be useful for fine structure genetic mapping. The goal of this proposal is to understand how enzymes catalyze the repair of mismatched nucleotides in Saccharomyces cerevisiae. Associated goals are to understand how mismatch repair interacts with genetic recombination, how mismatch repair contributes to the fidelity of DNA replication and if defects in mismatch repair are responsible for inherited diseases. The following lines of experimentation will be carried out: l) The MSH2 gene (MutS homolog), which functions in gene conversion and mismatch repair, will continue to be studied to define the role of mismatch repair in genetic recombination and mutation avoidance. 2) Biochemical analysis of overproduced MSH2 protein will be continued to define its DNA binding properties and to identify interacting proteins. 3) Biochemical characterization of a S. cerevisiae in vitro mismatch repair system will he continued to identify and purify enzymes required for mismatch repair. 4) Genetic analysis of the MSH1 gene and biochemical analysis of the overproduced MSH1 protein will be continued to define the role MSH1 plays in maintaining the integrity of mitochondrial DNA. The ultimate goal of these experiments is to reconstitute a S. cerevisiae mismatch repair reaction with purified proteins and determine the mechanism of this reaction. In addition, it is anticipated that these studies will provide tools for use in the analysis of mismatch repair in higher eukaryotes.
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