Biochemical and Mechanistic Insights into the Roles of Rad51 Paralogs in Homologous Recombination Repair

Rad51 旁系同源物在同源重组修复中作用的生化和机制见解

• 批准号: 10450660
• 负责人: Jacob Garrett Thrasher
• 金额: $ 4.68万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10450660
• 关键词: BRCA1 gene; BRCA2 gene; Binding; Biochemical; Biological; Biological Assay; Cancer-Predisposing Gene; Cell Line; Cells; Code; Collaborations; Complex; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Repair Pathway; DNA biosynthesis; DNA replication fork; Data; Disease; Epithelial ovarian cancer; Etiology; Fanconi' s Anemia; Fellowship; Filament; Genetic study; Germ-Line Mutation; Hereditary Breast Carcinoma; Hereditary Breast and Ovarian Cancer Syndrome; Hereditary Malignant Neoplasm; Human; Individual; Inherited; Investigation; Kinetics; Knock-out; Knowledge; Link; Malignant Neoplasms; Malignant neoplasm of ovary; Mammalian Cell; Maps; Measures; Mediating; Mentors; Methods; Microscopy; Molecular; Mutate; Mutation; Mutation Analysis; Nonhomologous DNA End Joining; Nucleoproteins; PALB2 gene; Pathway interactions; Patients; Platinum; Play; Predisposition; Process; Protein Family; Proteins; Protocols documentation; RAD51C gene; Regulation; Research; Research Personnel; Research Training; Resolution; Role; Sampling; Scheme; Serous; Single-Stranded DNA; Site; Somatic Mutation; System; Techniques; Testing; TimeLine; Training; Work; XRCC2 gene; XRCC3 gene; base; brca gene; career; chemotherapy; experimental study; genome integrity; homologous recombination; in vivo; innovation; insight; malignant breast neoplasm; multidisciplinary; novel; paralogous gene; patient stratification; preservation; recombinational repair; reconstitution; recruit; repaired; resistance mechanism; response; spatial relationship; spatiotemporal; synergism; therapy resistant; treatment response; tumor

PROJECT SUMMARY/ABSTRACT Understanding the molecular pathways disrupted in breast and ovarian cancer is vital to combating these devastating diseases. Hereditary susceptibility to cancer can be caused by germline mutations in the BReast CAncer susceptibility genes BRCA1 and BRCA2. The BRCA genes code for proteins involved in homologous recombination (HR) repair of DNA Double-Stranded Breaks (DSBs) and the protection of reversed replication forks. BRCA2, in particular, is vital to HR as it loads RAD51 onto single-stranded DNA (ssDNA) forming a nucleoprotein filament. The five RAD51 paralogs: RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3 are a family of proteins with homology to RAD51, have been implicated in HR regulation, and germline mutations have been linked to familial breast and ovarian cancer. The RAD51 paralogs form two different complexes in human cells but their functions and stoichiometric relationships in HR remain uncharacterized decades after their discovery. The BCDX2 complex (RAD51B/C/D/XRCC2) and the CX3 complex (RAD51C and XRCC3) have yet to be successfully purified for biochemical assays. Genetic studies have made little progress as knockout cell lines for any one of the paralogs are not viable. This proposal will utilize novel methods to overcome these obstacles and better characterize the roles of the RAD51 paralogs in HR. The first approach will determine whether specific domains mediate an interaction between RAD51 paralog proteins and BRCA2 or PALB2 (Partner and Localizer of BRCA2). Second, the RAD51 paralogs will be purified from human cells using a unique and innovative protocol we have developed. The individual paralogs, as well as the two paralog complexes, will be tested to determine if synergy exists with BRCA2 in stimulating RAD51-mediated DNA strand exchange. In a complementary cell biological approach, I will use a conditional system to systematically deplete individual RAD51 paralogs, and their respective complexes, to observe if RAD51 foci formation is compromised in response to DNA damage. Finally, in collaboration with Dr. Eli Rothenberg at NYU, we will utilize his expertise in super-resolution microscopy to test the hypothesis that the RAD51 paralog complexes are recruited to DSBs to perform specific functions during HR. Using this technique, I will create a timeline of recruitment and retention at DSBs in the context of other HR proteins. This spatiotemporal information will inform us at which step in HR the paralogs are active and their spatial relationships at a single DSB. Upon completion of the research and training fellowship, the RAD51 paralogs will be significantly more understood, and the applicant will have received extensive training. The multidisciplinary mentoring team will prepare the applicant for research independence and a successful career as a cancer researcher.

项目摘要/摘要 了解乳腺癌和卵巢癌中被破坏的分子通路对于防治这些疾病至关重要。 毁灭性的疾病。遗传性癌症易感性可由乳房中的生殖系突变引起 癌症易感基因BRCA1和BRCA2。BRCA基因编码与同源基因相关的蛋白质 DNA双链断裂的重组修复和反向复制保护 叉子。BRCA2尤其对HR至关重要，因为它将RAD51装载到单链DNA(SsDNA)上，形成 核蛋白细丝。五个RAD51并行器：RAD51B、RAD51C、RAD51D、XRCC2和XRCC3是 与RAD51同源的蛋白质家族与HR调节有关，生殖系突变 与家族性乳腺癌和卵巢癌有关。RAD51蛋白在人体内形成两种不同的复合体 但它们的功能和HR中的化学计量关系在几十年后仍未确定 发现号。BCDX2复合体(RAD51B/C/D/XRCC2)和CX3复合体(RAD51C和XRCC3)尚未 成功地提纯后用于生化分析。作为基因敲除细胞的遗传学研究进展甚微 任何一条并列的线路都是不可行的。这项提议将利用新的方法来克服这些 并更好地描述了RAD51并列基因在人力资源中的作用。第一种方法将确定 特定结构域是否介导RAD51类蛋白与BRCA2或PALB2之间的相互作用 (BRCA2的合作伙伴和本地化)。其次，将使用一种独特的方法从人类细胞中提纯RAD51对映体 和我们开发的创新协议。单独的并列以及两个并列复合体将 进行测试，以确定在刺激RAD51介导的DNA链交换方面是否与BRCA2存在协同作用。在一个 补充细胞生物学的方法，我将使用条件系统来系统地耗尽个体 RAD51并列曲线及其各自的复合体，以观察RAD51病灶的形成是否受到损害 对DNA损伤的反应。最后，我们将与纽约大学的Eli Rothenberg博士合作，利用他的专业知识 在超分辨显微镜中测试RAD51副对数复合体被招募到DSB的假设 在人力资源期间执行特定的职能。使用这项技术，我将创建一个招聘和保留的时间表 在其他HR蛋白的背景下，DSB。这个时空信息将告诉我们在HR中处于哪个阶段 副鱼是活跃的，它们的空间关系在一个DSB上。在完成研究和 培训奖学金，RAD51 Paralog将得到更多的理解，申请者将拥有 接受了广泛的培训。多学科指导团队将为申请者的研究做好准备 独立性和作为癌症研究人员的成功职业生涯。