Repair of Oxidatively Damaged Guanines in Human

描述（由申请人提供）：DNA修复缺陷可导致基因组不稳定，这是癌症的标志。DNA氧化损伤是生物体突变负荷的主要来源，在致癌和衰老中起着重要作用。8-氧-7,8-二氢脱氧鸟嘌呤（8-oxoG）损伤是氧化损伤的主要稳定产物，由于它在DNA复制过程中可能与腺嘌呤错配，因此具有最有害的影响。因此，通过hMYH （MutY腺嘌呤糖基化酶同源物）、hOGG1（一种8-oxoG糖基化酶）和hMSH2/hMSH6依赖的错配修复途径修复ol A/8-oxoG和C/8-oxoG提供了对氧化应激的防御水平。本项目的总体目标是了解hMYH途径在控制癌变中的作用及其与其他修复途径的相互作用，以响应氧化应激，如过氧化氢和电离辐射处理。hMYH与复制蛋白hPCNA和hRPA、修复酶hAPE1和hMSH6以及细胞周期检查点蛋白hHusl/hRadl/hRad9的相互作用将被研究。(1)研究氧化应激下hMYH与hPCNA和hHusl相互作用的变化。我们将绘制hMYH和hHusl的相互作用结构域，并在体内测试它们的功能相互作用的意义。hMYH- hpcna与hMYH-hRad9/hRadl/hHusl之间发生分子开关以诱导DNA损伤反应，以及hMYH可能将hRad9/hRadl/hHusl招募到损伤部位的工作模型将进行测试。（II） hMYH和hMSH6的相互作用将在体内和体外得到阐明。将以裂变酵母S. pombe为模型系统，对hMYH突变体的视觉功能进行测试。（III） hMYH与hAPE 1、hPCNA、hHus 1、hRPA和hMSH6共定位的修复和复制位点将被检测。（IV）确定DNA损伤后hMYH的磷酸化状态，确定hMYH磷酸化的激酶，确定hMYH的磷酸化位点。hMYH活性与DNA损伤后蛋白质磷酸化或某些蛋白质-蛋白质相互作用的相关性将被检查。这项研究将促进我们对DNA修复在肿瘤易感性中的作用的理解。

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.