Recombinational Mechanisms of DNA Repair

DNA修复的重组机制

• 批准号: 10667606
• 负责人: Wolf-Dietrich Heyer
• 金额: $ 41.2万
• 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/10667606
• 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; Homologous Gene; Human; In Vitro; Invaded; Ionizing radiation; Lead; Length; Malignant Neoplasms; Malignant neoplasm of prostate; Maps; Measures; Mediating; Metabolism; Modality; Modeling; Molecular; Motor; Mutation; Nucleotides; Outcome; Pathway interactions; Poly(ADP-ribose) Polymerase Inhibitor; 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; Work; Yeasts; antitumor agent; 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.

项目摘要 同源重组（HR）通过高保真修复DNA双链维持基因组稳定性 断裂（DSB）和其他由普通抗肿瘤药物直接或间接诱导的复杂DNA损伤 包括电离辐射，拓扑异构酶靶向药物，链间交联药物以及引起的剂 复制叉停滞。人力资源缺陷对癌症具有双重意义，首先导致基因组不稳定性和 癌症易感性增加。此外，人力资源缺陷会导致特定的细胞漏洞 通过传统的基于DNA损伤的治疗或有针对性的治疗进行治疗的剥削 通过聚（ADP-核糖）聚合酶抑制。总体目标是阐明人力资源的机制。这 应用侧重于中央HR中间体，位移环（D-Loop），代表 人力资源子街道的分支点。具体目的是： AIM 1：定义细胞中的D环长度和RDH54在D环代谢中的作用 我们将采用生化（AIM 1A）和遗传方法（AIM 1B），以首次确定长度 使用新开发的测定法，该测定法以单分子分辨率映射D环。在AIM 1C中，我们将 确定RDH54在控制D环长度和跨界结果中的作用。 目标2：描述人Rad54b在人力资源中的作用 在测试与RAD54的功能合作模型时，我们将通过使用Rad54b在HR中的作用 一种生化方法，用于确定其在RAD51介导的重组中的作用，并使用已建立和新的 开发测定（AIM 2A）。在AIM 2B中，我们根据初步数据将这些研究扩展到人类细胞 显示了RAD54B在依赖于合成的链退火（SDSA）中的特定作用。在AIM 2C中，我们将适应 对人类细胞的新测定，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环是中央HR中间体和高度动态的。我们推测，D-Loops涵盖了一套多样的集合 解释了多个D环编辑途径的结构。基于令人兴奋的初步数据 我们将专注于RECQ1作为与SDSA中RECQ5共同表演的新颖玩家。我们将确定 SDSA中的机理RECQ1和RECQ5使用生化的体外重构方法（AIM 3A） AIM 3B中的遗传和细胞生物学方法补充。