Role of ATR in the DNA Damage Response

DNA损伤检查点是允许细胞监测基因组的完整性并向细胞发出DNA损伤存在的信号的细胞监视机制。这些途径在维持基因组稳定性方面起着关键作用，检查点功能的丧失与癌症的发展有关。基因组的不稳定性和某些检查点基因的缺陷也与导致过早衰老的几种综合征有关，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。本申请的重点是ATR（ATM和Rad 3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。主要目标是鉴定ATR和其他蛋白质中的结构域，这些结构域是ATR感知和识别细胞DNA损伤所需的。9-1-1复合物是ATR向其下游效应物发出信号所需的异源三聚体蛋白质复合物。本研究的具体目的是（1）确定9-1-1复合物是否是上游调节物和/或下游效应物。本研究的具体目的是（1）确定9-1-1复合物是否是ATR功能的上游调节器和/或下游效应器;（2）鉴定和表征ATR内的功能结构域;（3）纯化、克隆和表征介导ATR与DNA相互作用的蛋白质。非洲爪蟾卵提取物将用于研究9-1-1复合物与ATR之间的关系。使用我们开发的一系列测定，将在哺乳动物细胞中探测表现出独特的核定位和显性阴性表型的特定ATR片段的功能。最后，使用ATR的DNA结合活性的新测定和新的基于亲和力的纯化策略，将纯化、克隆和表征介导ATR与DNA相互作用的蛋白质。这些研究将提供一个更好的理解ATR信号的机制，他的存在不同形式的DNA损伤的细胞。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.