Regulation of RPA Activity in DNA Repair

描述（由申请人提供）：本提案的目的是了解异源三聚体人复制蛋白A（RPA;真核单链DNA结合蛋白）中间亚基（RPA 2）磷酸化的作用。从我的实验室产生的最新数据表明，RPA 2磷酸化提供了一个重要的调节控制细胞DNA损伤反应。使用的方法相结合，我们将在体内和体外的RPA磷酸化的作用的特点。具体来说，我们将研究的分子基础，其中低磷酸化RPA选择性地与复制叉，其相互作用的表征与MCM复合物。RPA与MCM的关联将被检查，无论是在溶液中，和合成的DNA复制叉结构。其次，使用RPA 2替换策略，我们将检查RPA 2磷酸化位点突变对重组介体BRCA 2和同源重组因子加载到DNA复制应激和DNA损伤位点的影响。将确定RPA 2突变对体外BRCA 2负载和体内HR速率的影响。第三，我们已经发现，这些激酶对RPA 2在遗传毒性应激条件下的协同活性调节随后的磷酸化事件在相同的RPA分子（顺式途径）和不同的RPA分子（反式途径）。通过探索顺式途径，我们将测试的假设，即S33磷酸化促进招聘的细胞周期蛋白A-Cdk 2的RPA基板在体外。为了研究反式反应，我们将研究RPA磷酸化对体内ATR动力学的影响。产生的数据将提供深入了解细胞周期位置如何导致DNA损伤被引导到不同的DNA修复途径。 公共卫生相关性：我们将了解RPA磷酸化在细胞对DNA损伤反应中的功能意义。这项工作的结果可能会导致新的治疗人类癌症的方法

DESCRIPTION (provided by applicant): The goal of this proposal is to understand the role of phosphorylation of the middle subunit (RPA2) of the heterotrimeric human replication protein A (RPA; the eukaryotic single-stranded DNA-binding protein). Recent data arising from my laboratory indicates that RPA2 phosphorylation provides an important regulatory control over the cellular DNA damage response. Using a combination of approaches, we will characterize the role of RPA phosphorylation in vivo and in vitro. Specifically, we will examine the molecular basis by which hypo-phosphorylated RPA selectively associates with the replication fork, by characterization of its interaction with the MCM complex. The association of RPA with MCM will be examined, both in solution, and on synthetic DNA replication fork structures. Second, using a RPA2 replacement strategy, we will examine the effect of RPA2 phosphorylation site mutation on the loading of the recombination mediator BRCA2 and homologous recombination factors to sites of DNA replication stress and DNA damage. The effects of RPA2 mutation on BRCA2 loading in vitro and on HR rates in vivo will be determined. Third, we have found that the synergistic activity of these kinases towards RPA2 under genotoxic stress conditions modulate subsequent phosphorylation events both on the same RPA molecule (the cis pathway) and on different RPA molecules (the trans pathway). By exploring the cis pathway, we will test the hypothesis that S33 phosphorylation facilitates recruitment of cyclin A-Cdk2 to the RPA substrate in vitro. To examine the trans reaction, we will examine the effect of RPA phosphorylation on ATR dynamics in vivo. The data generated will provide insights into how cell cycle position causes DNA lesions to be channeled into different DNA repair pathways. PUBLIC HEALTH RELEVANCE: We will understand the functional significance of RPA phosphorylation in the cellular response to DNA damage. The results from this work could lead to new threraputics to treat human cancers