Repair of Oxidatively Damaged Guanines in Human

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DESCRIPTION (provided by applicant): Defects in DNA repair can lead to genome instability, a hallmark of cancer. Oxidative DNA damage is a major source of mutation load in living organisms and plays a role in carcinogenesis and aging. The 8-oxo-7,8-dihydrodeoxyguanine (8-oxoG) lesion is a major stable product of oxidative damage and has the most deleterious effects because it can mispair with adenine during DNA replication. Thus, repair ol A/8-oxoG and C/8-oxoG by hMYH (MutY adenine glycosylase homolog), hOGG1 (an 8-oxoG glycosylase), and hMSH2/hMSH6 dependent mismatch repair pathways provide levels of defense against oxidative stress The overall goals of this project is to understand the role of hMYH pathway in controlling carcinogenesis and its interplay with other repair pathways in response to oxidative stress such as treatments by hydrogen peroxide and ionizing radiation. The interactions of hMYH with replication proteins hPCNA and hRPA, with repai] enzymes hAPE1 and hMSH6, as well as cell cycle checkpoint proteins hHusl/hRadl/hRad9 will be investigated. (I) Alterations in the interactions of hMYH with hPCNA and hHusl under oxidative stress will be investigated. The interacting domains on hMYH and hHusl will be mapped and the significance of their functional interaction will be tested in vivo. Working models that a molecular switch of hMYH-hPCNA tc hMYH-hRad9/hRadl/hHusl occurs to induce DNA damage response and that hMYH may recruit_ hRad9/hRadl/hHusl to the lesion sites will be tested. (II) Interaction between hMYH and hMSH6 will be elucidated both in vitro and in vivo. Fission yeast S. pombe will be used as a model system to test the in viw function of hMYH mutants. (III) Co-localization of hMYH with hAPE 1, hPCNA, hHus 1, hRPA, and hMSH6 ir repair and replication loci will be examined. (IV) The phosphorylation state of hMYH after DNA damage, the kinase responsible for the hMYH phosphorylation, and the phosphorylation site(s) of hMYH will be determined. The correlation of hMYH activities with protein phosphorylation or certain protein-protein interaction(s) after DNA damage will be examined. This study will advance our understanding of the role of DNA repair in tumor susceptibility.