Repair of Oxidatively Damaged Guanines

活性氧物种(ROS)通常在新陈代谢、反应过程中作为副产物产生 导致炎症，以及暴露在电离辐射和化学物质中。DNA氧化损伤呈现出一种 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ROS的DNA 损伤包括通过碱基切除修复(BER)修复的链断裂和氧化碱基损伤， 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MutY同源(HMYH)糖基酶的作用及其与其他修复机制的相互作用。 HMYH通过去除与8-oxoG(8-oxoG)配对的错误结合的腺嘌呤来减少应激诱导的突变( 最丰富的DNA损伤形式)，因此减少G：C到T：A突变。HMYH缺乏易感 个人对结肠癌的风险。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)和参与衰老调控的SIRT6。在……里面 在此背景下，9-1-1蛋白与hMYH糖基酶，hNEIL1糖基酶， HOGG1糖基酶和hMSH2/hMSH6错配识别复合体。我们假设MYH和其他人 DNA修复酶作为分子适配器，将检查点蛋白招募到DNA损伤部位和 协调DNA修复，提高修复效率和保真度。为了检验这一假设，我们提出了 具体目标如下：(1)MYH与OGG1、NEIL1和MSH2/MSH6的动态相互作用 体外和体内将被描绘出来。我们将测试这些相互作用在氧化后是否会改变 应激和在细胞周期的进程中。(2)MYH的物理和功能相互作用(和 NeIL1、hOGG1和hMSH2/hMSH6)与9-1-1复合体结合。我们将调查不同之处 蛋白质竞争9-1-1复合体以及为什么不同的糖基酶选择9-1-1的不同亚基 很复杂。Hus1-MYH相互作用的生物学意义将通过中断 通过诱变和使用Hus1结合的竞争多肽进行相互作用。我们将测试一种DNA模型 糖基酶充当适配器，将9-1-1复合体招募到病变部位。(3)老化调节器的作用 SIRT6通过刺激MYH而在误码率中发挥作用，但抑制NEIL1的活性将被研究。因为SIRT6需要 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很重要。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的相互作用。我们期待着 这些研究将促进我们对癌症发生过程的了解，并为 抗癌新药的开发。

Reactive oxygen species (ROS) are commonly generated as by-products during metabolism, during response to inflammation, and following exposure to ionizing radiation and chemicals. Oxidative DNA damage presents a serious challenge to genomic integrity and can accelerate carcinogenesis and aging. ROS-dependent DNA damage includes strand breaks and oxidative base lesions that 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 other repair mechanisms. 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 SIRT6 which is implicated in regulation of aging. In this context, the 9-1-1 proteins interact with and increase the activity of hMYH glycosylase, hNEIL1 glycosylase, hOGG1 glycosylase, 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 the following specific aims: (1) The dynamic interactions of MYH with OGG1, NEIL1, and 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 role of an aging regulator SIRT6 in BER by stimulating MYH, but inhibiting NEIL1 activity will be studied. Because SIRT6 is required to regulate genomic integrity and impacts the aging process, revealing the mechanism of SIRT6 interaction in BER 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. We anticipate that these studies will advance our understanding of carcinogenesis process and form the background work for the development of new anti-cancer drugs.