Mismatch repair is an error avoidance pathway devoted to enhancing the fidelity of DNA replication and maintaining genetic stability. Mutations in human mismatch repair genes predispose individuals to cancer. Oxidative stress and metabolic processes which produce reactive oxygen species have been implicated as important causative agents of mutagenesis, carcinogenesis, aging, and a number of diseases. The human MutY homolog (hMYH) mismatch repair pathway for repairing A/G, A/C, and A/8-oxoG mismatches will be our major focus. The 8-oxoG lesion is a major stable product of DNA oxidative damage and has the most deleterious effects because it can mispair with adenine. Thus, A/8-oxoG mismatches are particularly important biological substrates for hMYH. hMYH, like the E. coli MutY protein, is an adenine DNA glycosylase. Because recombinant hMYH protein expressed in E. coli and native hMYH have different mismatch specificities, the structural and functional differences of these proteins will be further analyzed. Glycosylase and apurinic/apyrimidinic (AP) lyase activities on DNA containing A/G, A/C, A/8-oxoG, and other base analogs will be assayed. Our results indicate the hMYH, MutS homologs (hMSH2 and hMSH6), and MutL homologs (hMLH1 and hPMS2) are associated with the DNA replication complex, thus the interactions between hMYH and replicative as well as mismatch repair proteins will be investigated by co-immunoprecipretation and affinity chromatography. The coupling of hMYH repair with DNA replication may direct MYH repair to the misincorporated adenines on the daughter strands. The effect of proliferating cell nuclear antigen (PCNA, an accessory factor for DNA polymerases delta and epsilon) on hMYH activity will be determined. Human breast and lung cancer cells will be analyzed for hMYH expression and screened for mutations in the hMYH gene. The sensitivities of the MYH defective cells to oxidative agents and radiation will be analyzed. Through the study of the mechanism of DNA mismatch repair, our understanding of cancer, aging, and genetic diseases can be advanced.
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