Regulation of RPA Activity in DNA Repair

描述(由申请人提供)：本提案的目标是了解异源三聚体人类复制蛋白A(RPA；真核单链DNA结合蛋白)中间亚单位(RPA2)的磷酸化作用。最近来自我的实验室的数据表明，RPA2的磷酸化对细胞DNA损伤反应提供了重要的调控。结合多种方法，我们将研究RPA磷酸化在体内和体外的作用。具体地说，我们将通过表征低磷酸化的RPA与MCM复合体的相互作用，来研究其选择性与复制叉结合的分子基础。RPA与MCM的关联将在溶液中和合成DNA复制叉状结构上进行检查。其次，使用RPA2替换策略，我们将检测RPA2磷酸化位点突变对重组介体BRCA2和同源重组因子在DNA复制应激和DNA损伤部位的负载的影响。RPA2突变对BRCA2体外负荷和体内心率的影响将被确定。第三，我们发现，在基因毒性应激条件下，这些激酶对RPA2的协同活性调节了随后在同一RPA分子(顺式途径)和不同RPA分子(反式途径)上的磷酸化事件。通过探索顺式途径，我们将检验S33磷酸化促进细胞周期蛋白A-CDK2在体外募集到RPA底物的假说。为了检验反式反应，我们将检测RPA磷酸化对体内ATR动力学的影响。产生的数据将提供关于细胞周期位置如何导致DNA损伤被引导到不同的DNA修复途径的见解。 公共卫生相关性：我们将了解RPA磷酸化在细胞对DNA损伤的反应中的功能意义。这项工作的结果可能会导致新的治疗人类癌症的药物

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