Cellular Responses to UV Light in Ataxia Telangiectasia

描述(申请人提供)：我们建议研究DNA损伤诱导的、ATM/ATR依赖的主要细胞单链DNA结合蛋白RPA的磷酸化的功能意义。由于RPA对DNA复制和DNA修复是必不可少的，依赖于磷酸化的RPA功能改变有可能改变细胞对DNA损伤的反应。事实上，我们已经证明了DNA损伤诱导的ATM依赖的RPA磷酸化发生，并在体外改变了RPA的功能。具体地说，RPA磷酸化改变了DNA结合活性和蛋白质/蛋白质相互作用。我们推测，在ATM突变体中，未能磷酸化RPA和DNA复制和DNA修复所需的其他蛋白质，会损害细胞从DNA复制到DNA修复的转换，而DNA修复通常会保护细胞免受DNA损伤的有害后果。我们认为，A-T的这些异常反应导致了与疾病相关的细胞死亡和遗传不稳定性的增强。我们建议检验这样一种假设，即未能磷酸化RPA(和其他DNA修复蛋白)以响应DNA损伤会损害A-T细胞的DNA修复能力。该项目的具体目标是： 1.确定ATM和ATR介导的RPA磷酸化的位点，并构建位点特异性突变体，以检测它们在RPA功能中的作用。 2.测定RPA(和突变型RPA)磷酸化对体内和体外蛋白质相互作用的影响。一种新开发的RPA磷酸化特异性抗体将极大地促进这些研究。 3.要阐明ATM激酶活性与其他ATM功能在DSB修复中的作用： 我们正在集中研究A-T细胞对DNA损伤的异常反应，这种反应可能会影响这些细胞的DNA修复能力，并可能导致高癌症风险和神经变性。这个项目的最终目标是提高我们对细胞用来维持基因组稳定性的机制的理解。这种更好的理解最终可能导致制定预防和/或治疗以基因组不稳定和神经缺陷为特征的疾病的策略。

DESCRIPTION (provided by applicant): We propose to investigate the functional significance of DNA-damage-induced, ATM/ATR-dependent phosphoryIation of the major cellular single-stranded DNA-binding protein, RPA. Since RPA is essential for DNA replication and DNA repair, phosphorylation-dependent alteration in RPA function has the potential of altering the cellular responses to DNA damage. Indeed, we have demonstrated that DNA damage-induced ATM-dependent phosphorylation of RPA occurs, and it changes RPA function in vitro. Specifically, RPA phosphorylation alters DNA binding activity and protein/protein interactions. We postulate that a failure to phosphorylate RPA and other proteins required for DNA replication and DNA repair in ATM mutants compromises the cellular switch from DNA replication to DNA repair that would normally protect cells from the deleterious consequences of DNA damage. We propose that these abnormal responses in A-T lead to the enhanced cell death and genetic instability associated with the disease. We propose to test the hypothesis that the failure to phosphorvlate RPA (and other DNA repair proteins) in response to DNA damage compromises the DNA repair capacity of A-T ceils. The specific aims of this project are: 1. To determine the sites of ATM- and ATR-mediated RPA phosphorylation and construct site-specific mutants to test their role in RPA function. 2. To determine the influence of RPA (and mutant RPA) phosphorylation on protein/protein interactions in vivo and in vitro. A newly-developed RPA phospho-specific antibody will greatly facilitate these studies. 3. To clarify the role of the ATM kinase activity vs. other ATM functions in DSB repair: We are concentrating on abnormal responses to DNA damage in A-T cells that may influence the DNA repair capacity of these cells and may contribute to the high cancer risk and neurodegeneration. The ultimate goal of this project is to improve our understanding of the mechanisms that cells use to maintain genomic stability. This improved understanding may ultimately lead to the development of strategies for preventing and/or treating diseases that are characterized by genomic instability and neurological defects.