Dynamics of BRCA1-Mediated Double-Strand Break Repair

项目摘要 多种DNA损伤导致DNA双链断裂（DSB）的形成，无论是直接的还是间接的， 修复的中间体。为了对抗DNA损伤的积累，真核细胞采用了一种复杂的 促进损伤识别、检查点信号传导和DNA修复的途径网络。的组件 DNA损伤反应网络与各种遗传疾病有关，其典型表现为 对DNA损伤剂的超敏反应和癌症易感性。尤其是乳腺癌 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 破坏反应的各个方面。该提案旨在了解BRCA1如何调节 同源重组（HR）以促进DSB的无错修复。在S相中，DSB的末端是 由切除机械加工，以促进HR介导的修复，这利用了新的 复制的姐妹染色单体作为无错误修复的模板。在最近证实了非洲爪蟾卵 提取物可以重现DSB的重组依赖性修复，该系统将提供一个强大的工具， 为了研究BRCA1介导的HR的动力学事件， 将建立支持通过单分子成像进行分析的系统。新技术已经 开发支持实时成像在高度集中的爪蟾卵提取物，提供了一个显着的 与依赖于分离研究的纯化组分的传统单分子方法相比，该方法具有优势。 单分子成像将用于真实的分析BRCA 1依赖性DSB修复，提供一个水平 传统的集成方法无法提供的机械洞察力。通过这种方式， BRCA1可以被解剖，以确定细胞如何调节DNA损伤反应的不同方面。

Project Summary 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.