Repair of Oxidatively Damaged Guanines

描述（由申请人提供）： 氧化性DNA损伤对基因组完整性提出了严重挑战，并可加速致癌和衰老。活性氧依赖性DNA损伤通过碱基切除修复（BER）修复，起始于DNA糖基化酶去除碱基损伤。该项目的总体目标是研究选定的DNA糖基化酶和错配修复酶在细胞氧化应激反应中的作用。我们专注于人类MutY同源物（hMYH）糖基化酶的作用及其与细胞周期和衰老调节因子的相互作用。hMYH通过去除与8-oxoG（最丰富的DNA损伤形式）配对的错误掺入的腺嘌呤来减少应激诱导的诱变，因此减少G：C至T：A突变。hMYH缺乏使个体易患结肠癌。hMYH与DNA复制机制，其他修复酶，9-1-1细胞周期检查点复合物（Rad 9/Rad 1/Hus 1）和衰老调节因子SIRT 6相互作用。在这种情况下，9-1-1蛋白与DNA糖基化酶和hMSH 2/hMSH 6错配识别复合物相互作用并增加其活性。我们假设MYH和其他DNA修复酶作为分子衔接子，将检查点蛋白募集到DNA损伤位点，协调DNA修复，提高修复效率和保真度。为了验证这一假设，我们提出了三个具体的目标：（1）MYH与MSH 2/MSH 6的动态相互作用在体外和体内将被描绘。我们将测试这些相互作用是否在氧化应激后和细胞周期进展期间发生变化。(2)将阐明MYH（和Neil 1，hOGG 1和hMSH 2/hMSH 6）与9-1-1复合物的物理和功能相互作用。我们将研究不同的蛋白质如何竞争9-1-1复合物，以及为什么不同的糖基化酶选择9-1-1复合物的不同亚基。通过诱变和使用Hus 1结合竞争肽中断相互作用，研究Hus 1-MYH相互作用的生物学意义。我们将测试DNA糖基化酶作为衔接子将9-1-1复合物募集到病变部位的模型。(3)将研究衰老调节因子SIRT 6在DNA修复、细胞周期控制和衰老中的新作用。SIRT 6与9-1-1复合物相互作用，并通过刺激MYH但抑制NEIL 1活性在BER中发挥作用。我们将测试DNA糖基化酶的表达是否可以影响Sirt 6缺陷细胞对DNA损伤剂的敏感性，以及SIRT 6是否可以使hNEIL 1去乙酰化。由于SIRT 6是调节基因组完整性和影响衰老过程所必需的，因此揭示SIRT 6在BER和细胞周期检查点中的相互作用机制是重要的。这些研究的成功完成将揭示关于DNA修复蛋白、细胞周期检查点和衰老调节蛋白之间相互作用的重要新信息。这些研究将进一步加深我们对肿瘤发生过程的理解，并为开发新的抗癌药物奠定基础。 公共卫生相关性： 该项目的总体目标是解决几种DNA糖基化酶和错配修复酶在癌症预防中的作用。DNA修复、细胞周期检查点激活和基因沉默子SIRT 6的缺陷与癌症和衰老有关。我们的研究结果将对治疗人类疾病和癌症产生影响。

DESCRIPTION (provided by applicant): Oxidative DNA damage presents a serious challenge to genomic integrity and can accelerate carcinogenesis and aging. Reactive oxygen species-dependent DNA damage are repaired by base excision repair (BER), initiated with removal of base lesions by DNA glycosylases. The overall goal of this project is to study the role of selected DNA glycosylases and mismatch repair enzymes in response to cellular oxidative stress. We focus on the role of human MutY homolog (hMYH) glycosylase and its interactions with cell cycle and aging regulators. hMYH reduces stress-induced mutagenesis by removing misincorporated adenines paired with 8-oxoG (the most abundant form of DNA damage), therefore reduces G:C to T:A mutations. hMYH deficiency predispose individuals to colon cancer. hMYH interacts with the DNA replication machinery, other repair enzymes, the 9-1-1 cell cycle checkpoint complex (Rad9/Rad1/Hus1), and the aging regulator SIRT6. In this context, the 9-1-1 proteins interact with and increase the activities DNA glycosylases and hMSH2/hMSH6 mismatch recognition complex. We hypothesize that MYH and other DNA repair enzymes serve as molecular adaptors to recruit checkpoint proteins to DNA lesion sites and coordinate DNA repair and increase repair efficiency and fidelity. To examine this hypothesis, we propose three specific aims: (1) The dynamic interaction of MYH with MSH2/MSH6 both in vitro and in vivo will be delineated. We will test whether these interactions are altered following oxidative stress and during the progression of the cell cycle. (2) The physical and functional Interactions of MYH (and Neil1, hOGG1 and hMSH2/hMSH6) with the 9-1-1 complex will be elucidated. We will investigate how different proteins compete for the 9-1-1 complex and why different glycosylases select different subunits of the 9-1-1 complex. The biological significance of Hus1-MYH interaction will be investigated by interruption of the interaction by mutagenesis and by using a Hus1 binding competitor peptide. We will test a model that DNA glycosylases act as adaptors to recruit the 9-1-1 complex to the lesion sites. (3) The novel role of an aging regulator SIRT6 in DNA repair, cell cycle control, and aging will be studied. SIRT6 interacts with the 9-1-1 complex and has a role in BER by stimulating MYH, but inhibiting NEIL1 activity. We will test whether expression of DNA glycosylase can influence the sensitivity of Sirt6 deficient cells to DNA damage agents and whether SIRT6 can deacetylate hNEIL1. Because SIRT6 is required to regulate genomic integrity and impacts the aging process, revealing the mechanism of SIRT6 interaction in BER and cell cycle checkpoints is important. Successful completion of these studies will reveal important new information regarding the interactions among DNA repair proteins, cell cycle checkpoints, and an aging regulating protein. These studies will advance our understanding of carcinogenesis process and form the background work for the development of new anti-cancer drugs. PUBLIC HEALTH RELEVANCE: The overall goal of this project is to address the roles of several DNA glycosylases and mismatch repair enzymes in cancer prevention. Defects in DNA repair, cell cycle checkpoint activation, and gene silencer SIRT6 are associated with cancer and aging. The findings from our studies will have implications for treating human disease and cancer.