Recombinational Mechanisms of DNA Repair

DNA修复的重组机制

• 批准号: 10522982
• 负责人: Wolf-Dietrich Heyer
• 金额: $ 41.07万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10522982
• 关键词: ATRX gene; Abbreviations; Base Pairing; Biochemical; Biological; Biological Assay; Breast; Cells; Central Nervous System Neoplasms; Complement; Complex; Cruciform DNA; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Repair Pathway; DNA replication fork; DNA-Directed DNA Polymerase; Data; Defect; Double Strand Break Repair; Drug Targeting; Enzymes; Event; Funding; Genetic; Genetic Recombination; Genome Stability; Genomic Instability; Goals; Human; In Vitro; Ionizing radiation; Lead; Length; Light; Malignant Neoplasms; Malignant neoplasm of prostate; Maps; Measures; Mediating; Metabolism; Modality; Modeling; Molecular; Motor; Mutation; Nucleotides; Outcome; Pathway interactions; Poly(ADP-ribose) Polymerases; Process; Proteins; Quality Control; RAD54L gene; RECQL5 gene; Reaction; Regulation; Research; Resolution; Role; Saccharomyces cerevisiae; Saccharomycetales; Sister Chromatid Exchange; Structure; Technology; Testing; Therapeutic; Time; Topoisomerase; Ursidae Family; Work; Yeasts; antitumor agent; base; cancer predisposition; cancer therapy; cancer type; crosslink; genetic approach; homologous recombination; improved; insight; novel; novel therapeutic intervention; personalized medicine; reconstitution; single molecule; targeted treatment; tumor

Project Summary Homologous recombination (HR) maintains genomic stability through high-fidelity repair of DNA double-stranded breaks (DSB) and other complex DNA damage that is induced directly or indirectly by common anti-tumor agents including ionizing radiation, topoisomerase-targeted drugs, interstrand crosslinking agents, and those causing replication forks stalling. HR defects bear dual significance for cancer by first leading to genomic instability and increased cancer predisposition. Moreover, HR defects cause specific cellular vulnerabilities that can be exploited therapeutically either by traditional DNA damage-based treatment or by targeted treatment for example by poly(ADP-ribose) polymerase inhibition. The overarching goal is to elucidate the mechanisms of HR. This application focuses on a central HR intermediate, the displacement loop (D-loop), which represents the branchpoint for the HR sub-pathways. The Specific Aims are: Aim 1: Define D-loop length in cells and the role of Rdh54 in D-loop metabolism We will take biochemical (Aim 1A) and genetic approaches (Aim 1B) to determine for the first time the length of D-loops using a newly developed assay that maps D-loops at single molecule resolution. In Aim 1C, we will determine the role of Rdh54 in controlling D-loop length and crossover outcome. Aim 2: Delineate the role of human RAD54B in HR In testing the model of functional cooperation with RAD54, we will determine the role of RAD54B in HR by using a biochemical approach to determine its role in RAD51-mediated recombination using established and newly developed assays (Aim 2A). In Aim 2B, we extend these studies to human cells based on preliminary data showing a specific role of RAD54B in synthesis-dependent strand annealing (SDSA). In Aim 2C, we will adapt novel assays to human cells, determine D-loop length, and test the effect of RAD54B on D-loop levels and length to determine the mechanisms of crossover avoidance. Aim 3: Determine the roles of RECQ1 and RECQ5 in D-loop editing and crossover control D-loops are a central HR intermediate and highly dynamic. We surmise that D-loops encompass a diverse set of structures explaining the existence of multiple D-loop editing pathways. Based on exciting preliminary data, we will focus on RECQ1 as a novel player acting in concert with RECQ5 in SDSA. We will determine the mechanisms RECQ1 and RECQ5 in SDSA using a biochemical in vitro reconstitution approach (Aim 3A) complemented by genetic and cell biological approaches in Aim 3B.

项目概要 同源重组 (HR) 通过 DNA 双链的高保真修复维持基因组稳定性 常见抗肿瘤药物直接或间接诱导的断裂 (DSB) 和其他复杂的 DNA 损伤 包括电离辐射、拓扑异构酶靶向药物、链间交联剂以及那些引起 复制叉停止。 HR 缺陷对癌症具有双重意义，首先导致基因组不稳定， 增加癌症易感性。此外，人力资源缺陷会导致特定的细胞脆弱性，这些脆弱性可以 例如，通过传统的基于 DNA 损伤的治疗或靶向治疗来进行治疗 通过聚（ADP-核糖）聚合酶抑制。首要目标是阐明人力资源机制。这 应用重点关注中心 HR 中间体，即位移环（D 环），它代表 HR 子路径的分支点。具体目标是： 目标 1：定义细胞中 D 环长度以及 Rdh54 在 D 环代谢中的作用 我们将采用生化（目标 1A）和遗传方法（目标 1B）来首次确定 D 环使用新开发的检测方法，以单分子分辨率绘制 D 环图谱。在目标 1C 中，我们将 确定 Rdh54 在控制 D 环长度和交叉结果中的作用。 目标 2：描述人类 RAD54B 在 HR 中的作用 在测试与 RAD54 的功能合作模型时，我们将通过使用确定 RAD54B 在 HR 中的作用 一种生化方法，使用已建立的和新的方法来确定其在 RAD51 介导的重组中的作用 开发的测定（目标 2A）。在目标 2B 中，我们根据初步数据将这些研究扩展到人类细胞 显示 RAD54B 在合成依赖性链退火 (SDSA) 中的特定作用。在《Aim 2C》中，我们将进行调整 针对人体细胞的新颖测定法，确定 D 环长度，并测试 RAD54B 对 D 环水平和长度的影响 确定避免交叉的机制。 目标 3：确定 RECQ1 和 RECQ5 在 D 环编辑和交叉控制中的作用 D 环是 HR 的中心中间体，具有高度动态性。我们推测 D 环包含不同的集合 解释多个 D 环编辑途径存在的结构。基于令人兴奋的初步数据， 我们将重点关注 RECQ1 作为与 SDSA 中的 RECQ5 协同行动的新颖参与者。我们将确定 使用生化体外重构方法研究 SDSA 中的 RECQ1 和 RECQ5 机制（目标 3A） 通过 Aim 3B 中的遗传和细胞生物学方法进行补充。