Cellular Responses to UV Light in Ataxia Telangiectasia

描述（由申请人提供）：我们建议研究dna损伤诱导的主要细胞单链dna结合蛋白RPA的ATM/ atr依赖性磷酸化的功能意义。由于RPA对DNA复制和DNA修复至关重要，磷酸化依赖性的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依赖性RPA磷酸化发生，并且它改变了体外RPA功能。具体来说，RPA磷酸化改变了DNA结合活性和蛋白/蛋白相互作用。我们假设，在ATM突变体中，RPA和DNA复制和DNA修复所需的其他蛋白质的磷酸化失败会破坏细胞从DNA复制到DNA修复的转换，而这种转换通常会保护细胞免受DNA损伤的有害后果。我们认为这些在A-T中的异常反应导致与疾病相关的细胞死亡和遗传不稳定的增强。我们建议验证这一假设，即在DNA损伤的反应中，RPA（和其他DNA修复蛋白）磷酸化失败会损害A-T细胞的DNA修复能力。该项目的具体目标是：

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.