My long-term research goals are to study protein-DNA interactions and to understand molecular mechanisms of DNA mismatch repair. Mismatch repair is an error avoidance pathway devoted to enhancing the fidelity of DNA replication. Elimination of replicative errors is central to our understanding of the maintenance of genetic stability. One specific pathway in both E. coli and eukaryotes for repairing A/G and/or A/C mismatches will be our major focus. The E. coli MutY protein, involved in this pathway, is an iron-sulfur protein that has been shown to have both DNA glycosylase and 3' apurinic/apyrimidinic (AP) endonuclease activities. The MutY glycosylase can remove adenines from A/G, A/C, and A/8-oxoG mispairs. The 8-oxoG lesion is one of the most stable products of oxidative damage to DNA. Oxidative stress has been implicated as an important causative agent of mutagenesis, carcinogenesis, aging, and a number of diseases. One of my objectives is to study how MutY recognizes the mismatched DNA substrates. The bases and phosphates of DNA involved in the interaction have been determined by alkylation interference. The mutY gene will be mutated to identify which amino-acid residues are important in the MutY functions. Domains of MutY generated by proteolysis or expression of polymerase chain reaction (PCR) products will be tested for retention of iron-sulfur cluster binding, DNA substrate binding, glycosylase, and AP endonuclease activities. The defined protein structure will be studied by X-ray crystallography of MutY protein and MutY-DNA co-crystal. Endonuclease L that cuts at the 5' side of the mismatched adenines after MutY and other proteins involved in the E. coli MutY pathway will be purified and characterized. My mismatch repair research will extend into eukaryotes, including yeast and mammalian systems. Mismatch-specific nicking enzymes will be purified from calf thymus and human HeLa cells. Human cDNA for an A/G-specific enzyme will be cloned by probing with oligonucleotides deduced from the amino acid sequence or with antibodies against E. coli MutY. The all-type endonuclease, which can nick all the mismatched bases, will be purified from calf thymus and HeLa cells and compared to topoisomerase because their reactions are mechanistically similar. In order to construct yeast mutants defective in the A/G-specific enzyme for genetic studies, we will clone the yeast gene that is homologous to E. coli mutY. Through the study of the mechanism of DNA mismatch repair, mutagenesis, aging, and genetic diseases can be elucidated on a molecular level.
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