Role of ATR in the DNA Damage Response

DNA损伤检查点是一种细胞监测机制，允许细胞监控基因组的完整性，并发出DNA损伤对细胞存在的信号。这些通路在维持基因组稳定性方面起着关键作用，而检查点功能的丧失与癌症的发展有关。基因组不稳定和一些检查点基因的缺陷也与几种导致过早衰老的综合征有关，包括沃纳综合征。这项研究的长期目标是了解细胞通过信号传递不同形式的DNA损伤对细胞周期机制和DNA修复蛋白的影响的机制。这一应用的重点是ATR(ATM和Rad3相关)，这是一种哺乳动物基因，是DNA损伤和复制检查点所必需的。主要目标是确定ATR和其他蛋白质中的结构域，这些结构域是ATR检测和转导细胞DNA损伤的存在所必需的。9-1-1复合体是一种异三聚体蛋白质复合体，ATR可能需要它来向下游效应器发出信号。本研究的具体目的是(1)确定9-1-1复合体是否是上游调节因子和/或下游效应因子。本研究的具体目的是(1)确定9-1-1复合体是否是ATR功能的上游调节因子和/或下游效应因子；(2)鉴定和鉴定ATR内的功能结构域；(3)纯化、克隆和鉴定介导ATR与DNA相互作用的蛋白质。非洲爪哇卵的提取物将被用来研究9-1-1复合体与ATR的关系。利用我们开发的一系列分析方法，将探索特定的ATR片段在哺乳动物细胞中的功能，该片段具有独特的核定位和显性的负表型。最后，使用一种新的ATR与DNA结合活性的检测方法和一种新的基于亲和力的纯化策略，将对介导ATR与DNA相互作用的蛋白质进行纯化、克隆和鉴定。这些研究将更好地理解ATR向细胞发出不同形式的DNA损伤信号的机制。此外，它们应该为人类疾病，如癌症和复杂的人类老龄化过程提供重要的见解。

The DNA damage checkpoints are cellular surveillance mechanisms that allow the cell to monitor the integrity of the genome and to signal the presence of DNA damage to the cell. These pathways play a critical role in maintaining genomic stability, and loss of checkpoint function has been implicated in the development of cancer. Genomic instability and defects in some checkpoint genes have also been linked to several syndromes that lead to premature aging, including Werner's syndrome. The long-term objective of this research is to understand the mechanism by which cells signal the presence of different forms of DNA damage to the cell cycle machinery and DNA repair proteins. The focus of this application is ATR (ATM and Rad3-related), a mammalian gene that is required for the DNA damage and replication checkpoints. The primary goal is to identify domains in ATR and other proteins that are required for ATR to sense and transduce the presence of DNA damage to the cell. The 9-1-1 complex is a heterotrimeric protein complex that may be required for ATR to signal to its downstream effectors. The specific aims of this research are (1) to determine if the 9-1-1 complex is an upstream regulator and/or downstream effectors. The specific aims of this research are (1) to determine if the 9-1-1 complex is an upstream regulator and/or downstream effector of ATR function (2) to identify and characterize functional domains within ATR, and (3) to purify, clone and characterize the protein that mediates the interaction of ATR with DNA. Extracts from the eggs of Xenopus laevis will be used to study the relationship between the 9-1-1 complex and ATR. Using a series of assays that we developed, the function of a specific ATR fragment that exhibits a unique nuclear localization and a dominant negative phenotype will be probed in mammalian cells. Finally, using a new assay for ATR's DNA- binding activity and a new affinity-based purification strategy, the protein that mediates the interaction of ATR with DNA will be purified, cloned and characterized. The studies will provide a better understanding of the mechanisms by which ATR signals he presence of different forms of DNA damage to the cell. Furthermore, they should provide important insights into human disease such as cancer and the complex process of human aging.