Replication-Associated Repair and Replication Fork Maintenance

复制相关修复和复制叉维护

• 批准号: 8555254
• 负责人: Priscilla K. Cooper
• 金额: $ 29.19万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-27 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8555254
• 关键词: Abnormal Cell; Aging; Binding; Biochemical; Biological; Catalytic DNA; Catalytic Domain; Cell Cycle; Cells; Cellular biology; Collaborations; Complex; DNA; DNA Binding; DNA Repair; DNA Repair Pathway; DNA biosynthesis; DNA ligase I; DNA-Protein Interaction; Data; Disease; Distal; Double Strand Break Repair; Excision Repair; Failure; Funding; Genome; Genome Stability; Genomics; Goals; Lead; Lesion; Ligase; Maintenance; Malignant Neoplasms; Maps; Molecular; Mutation; Outcome Study; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphotransferases; Process; Property; Proteins; RTH-1 Nuclease; Reaction; Research; Resolution; Resources; Role; Source; Specificity; Stress; Structure; Substrate Domain; Testing; Therapeutic Agents; Therapeutic Intervention; Translating; XRCC1 gene; base; cancer cell; cancer therapy; endonuclease; helicase; inhibitor/antagonist; novel; programs; protein complex; repaired; small molecule; structural biology

Project 2 (Replication-Associated Repair and Replication Fork Maintenance) of the SBDR Program Project focuses on the integration of multiple DNA repair pathways at replication forks and their roles in the maintenance of genomic stability. Cells devote significant resources to detecting and repairing DNA damage prior to replication and to protecting active replication forks in the presence of replisome-stalling lesions. Failure in these processes or in their coordination can lead to cancer and aging. The goal of this research is to use a combination of structural and functional approaches to investigate the protein-protein and protein- DNA interfaces required for coordinated damage recognition and repair in association with replication. We propose four Aims to examine keystone proteins involved in excision repair pathways that remove damage prior to or in coordination with the replisome, and with proteins involved in fork stability. Aim 1 will investigate the structural and functional basis for roles of XPG in NER, BER, and replication-associated repair by atomic resolution studies of the structured endonuclease domain and by structural characterization of the relatively unstructured R- and C-terminus domains via their interaction with partner proteins RPA, ubiquitinated PCNA, and DNA Ligase I. Aim 2 will investigate the structural and functional basis for the role of the annealing helicase, SMARCAL1, with RPA at stalled replication forks. Since SMARCAL1 is the first annealing helicase demonstrated to act in maintaining genome integrity at stalled forks, it is critically important to understand its function at a mechanistic level. Aim 3 will investigate the structural biology of NEIL1-initiated BER of oxidized bases, through interactions with FEN-1 and XPG, XRCC1/Ligase III, and with RPA. Aim 4 will focus on PNKP, which has kinase and phosphatase activities critical for both single-strand and double-strand break repair processes and which is also an essential component of NEIL-directed BER. We will investigate PNKP phosphatase domain substrate binding, structurally characterize PNKP interaction with a specific inhibitor of its phosphatase activity, and interrogate the interaction of PNKP with XRCC1/Ligase III. The proposed studies are built upon major findings and collaborations generated during the previous funding period in SBDR2. They include experimental interactions and substantial synergy with Projects 1, 3, 4, and 6, as well as with the EMB and SCB Cores. The anticipated outcome of these studies is a much more detailed molecular picture of the protein-protein and protein-DNA complexes involved in replication-associated repair. The information generated will elucidate the mode of action of a promising DNA repair inhibitor and contribute to its optimization, and will define new potential targets for novel cancer therapies.

SBDR计划项目2（复制相关修复和复制叉维护）重点关注复制叉上多种DNA修复途径的整合及其在维持基因组稳定性中的作用。细胞投入大量资源在复制前检测和修复DNA损伤，并在存在复制体停滞病变时保护活跃的复制叉。如果这些过程或它们之间的协调出现故障，就会导致癌症和衰老。本研究的目的是利用结构和功能方法的结合来研究与复制相关的协调损伤识别和修复所需的蛋白质-蛋白质和蛋白质- DNA界面。我们提出了四个目的来研究参与切除修复途径的关键蛋白，这些途径可以在复制体之前或与复制体协调去除损伤，以及参与叉子稳定性的蛋白质。目的1将研究XPG在NER、BER和复制相关修复中的作用的结构和功能基础，通过对结构内切酶结构域的原子分辨率研究和相对非结构的R和c端结构域的结构表征，通过它们与伴侣蛋白RPA、泛素化PCNA和DNA连接酶i的相互作用。目的2将研究退火解旋酶SMARCAL1的作用的结构和功能基础。在停止复制分叉处使用RPA。由于SMARCAL1是第一个被证明在停滞分叉处维持基因组完整性的退火解旋酶，因此在机制水平上理解其功能至关重要。目的3将通过与FEN-1和XPG、XRCC1/连接酶III以及RPA的相互作用，研究neil1引发的氧化碱基BER的结构生物学。Aim 4将重点关注PNKP，它具有激酶和磷酸酶活性，对单链和双链断裂修复过程至关重要，也是neil定向BER的重要组成部分。我们将研究PNKP磷酸酶结构域底物结合，结构表征PNKP与特定磷酸酶活性抑制剂的相互作用，并询问PNKP与XRCC1/连接酶III的相互作用。