Role of ATR in the DNA Damage Response

DNA损伤检查点是细胞的监视机制，它允许细胞监测基因组的完整性，并向细胞发出DNA损伤的信号。这些途径在维持基因组稳定性方面发挥着关键作用，而检查点功能的丧失与癌症的发展有关。一些检查点基因的基因组不稳定和缺陷也与几种导致早衰的综合征有关，包括维尔纳综合征。本研究的长期目标是了解细胞对细胞周期机制和DNA修复蛋白发出不同形式DNA损伤信号的机制。该应用程序的重点是ATR （ATM和rad3相关），这是DNA损伤和复制检查点所必需的哺乳动物基因。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感知和转导细胞DNA损伤所必需的。9-1-1复合物是一种异三聚体蛋白复合物，ATR可能需要它向其下游效应物发出信号。本研究的具体目的是：(1)确定9-1-1复合物是上游调节剂和/或下游效应器。本研究的具体目的是：(1)确定9-1-1复合物是否是ATR功能的上游调节剂和/或下游效应物(2)鉴定和表征ATR内的功能域，以及(3)纯化、克隆和表征介导ATR与DNA相互作用的蛋白质。利用非洲爪蟾卵提取液研究9-1-1复合物与ATR之间的关系。利用我们开发的一系列检测方法，将在哺乳动物细胞中探测具有独特核定位和显性阴性表型的特定ATR片段的功能。最后，利用一种新的ATR DNA结合活性测定方法和一种新的基于亲和的纯化策略，将对介导ATR与DNA相互作用的蛋白进行纯化、克隆和表征。这些研究将使我们更好地了解ATR如何向细胞发出不同形式的DNA损伤信号的机制。此外，它们应该为癌症等人类疾病和人类衰老的复杂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.