Repair of Oxidatively Damaged Guanines

描述(由申请人提供)： DNA氧化损伤对基因组的完整性提出了严重的挑战，并会加速癌症的发生和衰老。依赖于活性氧的DNA损伤可以通过碱基切除修复(BER)来修复，碱基切除修复是通过DNA糖基酶去除碱基损伤开始的。这个项目的总体目标是研究选定的DNA糖基酶和错配修复酶在细胞氧化应激反应中的作用。我们着重于人MutY同源糖基酶(HMYH)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)配对的错误结合的腺嘌呤来减少应激诱导的突变，从而减少G：C到T：A的突变。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体(Rad9/Rad1/Hus1)和衰老调节因子SIRT6相互作用。在此背景下，9-1-1蛋白与DNA糖基酶和hMSH2/hMSH6错配识别复合体相互作用并增强其活性。我们假设MYH和其他DNA修复酶作为分子适配器，将检查点蛋白招募到DNA损伤部位，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目标：(1)描述MYH与MSH2/MSH6在体外和体内的动态相互作用。我们将测试这些相互作用是否会在氧化应激之后和细胞周期的进程中发生变化。(2)阐明MYH(以及NEIL1、hOGG1和hMSH2/hMSH6)与9-1-1复合体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复合体，以及为什么不同的糖基酶选择9-1-1复合体的不同亚单位。HUS1-MYH相互作用的生物学意义将通过突变和使用HUS1结合的竞争肽来中断相互作用来研究。我们将测试一个模型，即DNA糖基酶作为适配器，将9-1-1复合体招募到病变部位。(3)研究衰老调节因子SIRT6在DNA修复、细胞周期调控和衰老中的新作用。SIRT6与9-1-1复合体相互作用，通过刺激MYH，抑制NEIL1活性而在BER中发挥作用。我们将测试DNA糖基酶的表达是否会影响SIRT6缺陷细胞对DNA损伤剂的敏感性，以及SIRT6是否可以去乙酰化hNEIL1。由于SIRT6需要调节基因组的完整性并影响衰老过程，因此揭示SIRT6在BER和细胞周期检查点的相互作用机制具有重要意义。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。这些研究将加深我们对肿瘤发生过程的了解，并为开发新的抗癌药物奠定基础。 公共卫生相关性： 这个项目的总体目标是研究几种DNA糖基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SIRT6的缺陷与癌症和衰老有关。我们的研究结果将对人类疾病和癌症的治疗产生影响。

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.