Dynamics of BRCA1-Mediated Double-Strand Break Repair

BRCA1 介导的双链断裂修复动力学

• 批准号: 8972309
• 负责人: David Thomas Long
• 金额: $ 24.9万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 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 2017-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8972309
• 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; repaired; research study; response; single molecule; tool; treatment strategy

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.

项目摘要 多种DNA损伤直接或间接地导致DNA双链断裂(DSB)的形成 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 促进损伤识别、检查点信号和DNA修复的路径网络。组件 DNA损伤反应网络已经与各种遗传疾病联系在一起，典型的是 对DNA损伤剂的超敏反应与癌症易感性。尤其是乳腺癌的肿瘤 抑制基因BRCA1被描述为基因组稳定性的主要调节者，因为它参与了 损害反应的各个方面。这项建议旨在了解BRCA1如何监管 同源重组(HR)促进双链断裂的无错误修复。在S阶段，DSB的结束是 由切除机器处理，以促进HR中介修复，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。最近证实非洲爪哇的卵 提取物可以概括重组依赖的DSB修复，这个系统将提供一个强大的工具 阐明BRCA1介导HR的机制。一种新的DSB修复方法HR的动态事件研究 将建立支持单分子成像分析的系统。新的技术已经出现 开发了支持在高度浓缩的非洲爪哇卵提取物中进行实时成像的产品，提供了显著的 与传统的单分子方法相比，这种方法具有优势，因为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，提供一个水平 这是传统的合奏方法所不具备的机械洞察力。在这种情况下，的复杂功能 可以解剖BRCA1来确定细胞如何调控DNA损伤反应的不同方面。