Dynamics of BRCA1-Mediated Double-Strand Break Repair

BRCA1 介导的双链断裂修复动力学

• 批准号: 8658109
• 负责人: David Thomas Long
• 金额: $ 2.23万
• 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 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8658109
• 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）：各种DNA损伤导致DNA双链断裂（DSB）的形成，直接或作为修复的中间体。为了对抗DNA损伤的积累，真核细胞采用复杂的通路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损伤剂的超敏反应和癌症易感性。特别是，乳腺癌肿瘤抑制因子BRCA1已被描述为基因组稳定性的主要调节因子，因为其参与损伤反应的各个方面。该提案旨在了解BRCA1如何调节同源重组（HR）以促进DSB的无错误修复。在S期，DSB的末端被切除机制处理以促进HR介导的修复，其利用新复制的姐妹染色单体作为无错误修复的模板。最近建立的爪蟾卵提取物可以重演的重组依赖性修复的DSB，该系统将提供一个强大的工具来阐明BRCA1介导的HR的机制。为了研究HR的动态事件，将建立一个新的DSB修复系统，支持通过单分子成像分析。新技术已经开发出来，支持实时成像在高度浓缩的非洲爪蟾卵提取物，提供了一个显着的优势，传统的单分子方法，依赖于纯化的成分研究隔离。单分子成像将被用于分析BRCA 1依赖性DSB修复在真实的时间，提供了一个机制的洞察力水平与传统的合奏方法不可用。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。