Dynamics of BRCA1-Mediated Double-Strand Break Repair

描述(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产物。为了对抗DNA损伤的积累，真核细胞利用一个复杂的路径网络来促进损伤识别、检查点信号和DNA修复。DNA损伤反应网络的组成部分与各种遗传疾病有关，这些疾病的典型特征是对DNA损伤剂超敏和癌症易感性。特别是，乳腺癌肿瘤抑制基因BRCA1被描述为基因组稳定性的主要调节因子，因为它参与了损伤反应的各个方面。本提案旨在了解BRCA1如何调节同源重组(HR)以促进DSB的无错误修复。在S阶段，DSB的末端被切除机器处理以促进HR介导的修复，该机制利用新复制的姐妹染色单体作为无错修复的模板。最近证实非洲爪哇卵提取物可以概括DSB的重组依赖性修复，该系统将为阐明BRCA1介导的HR的机制提供一个强有力的工具。为了研究HR的动态事件，将建立一种新的DSB修复系统，支持单分子成像分析。已开发的新技术支持对高度浓缩的非洲爪哇卵提取液进行实时成像，提供了比传统的单分子方法更大的优势，传统的单分子方法依赖于分离研究的纯化成分。单分子成像将用于实时分析依赖BRCA1的DSB修复，提供传统集成方法所不具备的机制洞察力。通过这种方式，可以解剖BRCA1的复杂功能，以确定细胞如何调控DNA损伤反应的不同方面。

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.