Dynamics of BRCA1-Mediated Double-Strand Break Repair

BRCA1 介导的双链断裂修复动力学

• 批准号: 8508577
• 负责人: David Thomas Long
• 金额: $ 9万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-02 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8508577
• 关键词: BRCA1 Mutation; BRCA1 gene; Bacteriophages; Biological Assay; Cells; Chromatin; Complex; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA analysis; DNA lesion; Development; Dissociation; Double Strand Break Repair; Eukaryotic Cell; Event; Excision; Foundations; Genetic Recombination; Genome Stability; Hereditary Breast Carcinoma; Hereditary Disease; Hypersensitivity; Image; Individual; Lead; Link; Malignant Neoplasms; Malignant neoplasm of ovary; Mechanics; Mediating; Mentors; Movement; Mutate; Pathway interactions; Patients; Phase; Play; Predisposition; Process; Proteins; Regulation; Role; S Phase; Signal Transduction; Sister Chromatid; Site; System; Techniques; Time; Tumor Suppressor Genes; Tumor Suppressor Proteins; Xenopus; clinically relevant; egg; homologous recombination; insight; malignant breast neoplasm; novel; public health relevance; repaired; research study; response; single molecule; tool; treatment strategy

DESCRIPTION (provided by applicant): A variety of DNA lesions lead to the formation of DNA double-strand breaks (DSBs), either directly or as intermediates of repair. To counter the accumulation of DNA damage, eukaryotic cells employ a complicated network of pathways that promote damage recognition, checkpoint signaling, and DNA repair. Components of the DNA damage response network have been linked to various genetic disorders that are typified by hypersensitivity to DNA damaging agents and cancer predisposition. In particular, the breast cancer tumor suppressor BRCA1 has been described as a master regulator of genome stability due to its involvement in various aspects of the damage response. This proposal seeks to understand how BRCA1 regulates homologous recombination (HR) to promote error-free repair of DSBs. In S phase, the ends of a DSB are processed by the resection machinery to promote HR-mediated repair, which takes advantage of the newly replicated sister chromatid as a template for error-free repair. Having recently established that Xenopus egg extracts can recapitulate recombination-dependent repair of a DSB, this system will provide a powerful tool to elucidate the mechanism of BRCA1-mediated HR. To study the dynamic events of HR, a novel DSB repair system will be established that supports analysis by single-molecule imaging. New techniques have been developed that support real-time imaging in highly concentrated Xenopus egg extracts, providing a significant advantage over traditional single-molecule approaches that rely on purified components studied in isolation. Single-molecule imaging will be used to analyze BRCA1-dependent DSB repair in real time, providing a level of mechanistic insight not available with traditional ensemble approaches. In this way, the complex functions of BRCA1 can be dissected to determine how cells regulate different aspects of the DNA damage response.

描述（由申请人提供）：多种 DNA 损伤直接或作为修复中间体导致 DNA 双链断裂 (DSB) 的形成。为了对抗 DNA 损伤的积累，真核细胞采用复杂的途径网络来促进损伤识别、检查点信号传导和 DNA 修复。 DNA 损伤反应网络的组成部分与各种遗传性疾病有关，这些遗传性疾病的典型特征是对 DNA 损伤剂过敏和癌症易感性。特别是，乳腺癌肿瘤抑制因子 BRCA1 因其参与损伤反应的各个方面而被描述为基因组稳定性的主要调节因子。该提案旨在了解 BRCA1 如何调节同源重组 (HR) 以促进 DSB 的无差错修复。在S期，DSB的末端被切除机器处理以促进HR介导的修复，其利用新复制的姐妹染色单体作为无差错修复的模板。最近确定非洲爪蟾卵提取物可以重现 DSB 的重组依赖性修复，该系统将为阐明 BRCA1 介导的 HR 机制提供强大的工具。为了研究 HR 的动态事件，将建立一种支持单分子成像分析的新型 DSB 修复系统。新技术已经开发出来，支持对高度浓缩的非洲爪蟾卵提取物进行实时成像，与依赖于分离研究的纯化成分的传统单分子方法相比，具有显着的优势。单分子成像将用于实时分析 BRCA1 依赖性 DSB 修复，提供传统整体方法无法提供的机制洞察。通过这种方式，可以剖析 BRCA1 的复杂功能，以确定细胞如何调节 DNA 损伤反应的不同方面。