Repair of Oxidatively Damaged Guanines

DNA氧化损伤是对基因组完整性的严重挑战，并可能加速癌症的发生 和衰老。依赖于活性氧的DNA损伤通过碱基切除修复(BER)来修复， 由DNA糖基酶去除碱基损伤开始。这个项目的总体目标是研究 选择的DNA糖基酶和错配修复酶对细胞氧化应激的反应。我们专注于 人MUY同源糖基酶的作用及其与细胞周期和衰老的相互作用 监管者。HMYH通过去除错误结合的腺嘌呤与8-甲基-2，4-二磷酸核苷结合来减少应激诱导的突变 因此，oxoG(最丰富的DNA损伤形式)会减少G：C到T：A的突变。HMYH缺乏症 易患结肠癌。HMYH与DNA复制机制相互作用，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。在这 在此背景下，9-1-1蛋白与DNA糖基酶和hMSH2/hMSH6相互作用并增强其活性 错配识别复合体。我们假设MYH和其他DNA修复酶作为分子 将检查点蛋白招募到DNA损伤部位并协调DNA修复和增加修复的接头 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)动态交互 将描述MYH与MSH2/MSH6在体外和体内的相互作用。我们将测试这些交互作用是否 在氧化应激后和细胞周期的进程中发生变化。(2)物质上的和 MYH(以及NEIL1、hOGG1和hMSH2/hMSH6)与9-1-1复合体的功能相互作用将是 已澄清。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 糖基酶选择9-1-1复合体的不同亚基。Hus1-MYH的生物学意义 交互作用将通过突变和使用Hus1结合来中断交互作用来研究 竞争者多肽。我们将测试DNA糖基酶作为适配器的模型，以招募9-1-1复合体 病变部位。(3)衰老调节剂SIRT6在DNA修复、细胞周期控制和衰老过程中的新作用 被研究。SIRT6与9-1-1复合体相互作用，通过刺激MYH但抑制MYH在误码率中发挥作用 NEIL1活性。我们将测试DNA糖基酶的表达是否会影响SIRT6的敏感性 缺陷细胞对DNA损伤剂的作用以及SIRT6是否能使hNEIL1去乙酰化。因为需要SIRT6 调节基因组完整性和影响衰老过程，揭示SIRT6相互作用的机制 BER和细胞周期检查点很重要。这些研究的成功完成将揭示出重要的新 关于DNA修复蛋白、细胞周期检查点和衰老之间相互作用的信息 调节蛋白质。这些研究将促进我们对癌症发生过程的理解，并形成 研发抗癌新药的背景工作。

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.