Regulation of RPA Activity in DNA Repair

DNA 修复中 RPA 活性的调节

• 批准号: 8035426
• 负责人: JAMES A. BOROWIEC
• 金额: $ 47.93万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8035426
• 关键词: BRCA2 gene; Binding; Cell Cycle; Cell Cycle Progression; Charge; Complex; Cultured Cells; Cyclin A; Cyclins; DNA; DNA Damage; DNA Repair; DNA Repair Pathway; DNA Replication Damage; DNA biosynthesis; DNA lesion; DNA replication fork; Data; Development; Event; Genetic Recombination; Genotoxic Stress; Goals; Human; In Vitro; Laboratories; Lead; Malignant Neoplasms; Mediator of activation protein; Mitosis; Mitotic; Modification; Molecular; Mutation; Pathway interactions; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Positioning Attribute; Proteins; Reaction; Recruitment Activity; Regulation; Reporting; Role; SS DNA BP; Site; Solutions; Stress; Structure; System; Testing; Tumor Suppressor Proteins; Variant; Work; base; biological adaptation to stress; design; experience; helicase; homologous recombination; in vivo; insight; mutant; public health relevance; recombinational repair; replication factor A; research study; response; synthetic construct

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

描述（由申请人提供）：本提案的目的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）的中间亚基（RPA2）磷酸化的作用。我实验室的最新数据表明 RPA2 磷酸化对细胞 DNA 损伤反应提供了重要的调节控制。通过结合多种方法，我们将描述 RPA 磷酸化在体内和体外的作用。具体来说，我们将通过表征低磷酸化 RPA 与 MCM 复合物的相互作用来检查低磷酸化 RPA 选择性与复制叉结合的分子基础。将在溶液中和合成 DNA 复制叉结构上检查 RPA 与 MCM 的关联。其次，使用 RPA2 替换策略，我们将检查 RPA2 磷酸化位点突变对重组​​介质 BRCA2 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 RPA2 突变对体外 BRCA2 负荷和体内 HR 率的影响。第三，我们发现这些激酶在基因毒性应激条件下对 RPA2 的协同活性调节相同 RPA 分子（顺式途径）和不同 RPA 分子（反式途径）上的后续磷酸化事件。通过探索顺式途径，我们将检验以下假设：S33 磷酸化促进细胞周期蛋白 A-Cdk2 在体外招募至 RPA 底物。为了检查反式反应，我们将检查 RPA 磷酸化对体内 ATR 动力学的影响。生成的数据将提供关于细胞周期位置如何导致 DNA 损伤被引导至不同 DNA 修复途径的见解。 公共健康相关性：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带来治疗人类癌症的新疗法