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

项目总结/摘要 了解乳腺癌和卵巢癌中被破坏的分子通路对于对抗这些疾病至关重要。 毁灭性的疾病遗传性癌症易感性可由乳腺癌的生殖系突变引起。 癌症易感基因BRCA 1和BRCA 2。BRCA基因编码的蛋白质参与同源 DNA双链断裂（DSB）的重组（HR）修复和反向复制的保护 叉子BRCA 2对HR尤其重要，因为它将RAD 51装载到单链DNA（ssDNA）上，形成一个 核蛋白丝RAD 51的五个旁系同源物：RAD 51 B，RAD 51 C，RAD 51 D，XRCC 2和XRCC 3是一个 与RAD 51具有同源性的蛋白质家族与HR调节有关，种系突变也与HR调节有关。 与家族性乳腺癌和卵巢癌有关。RAD 51旁系同源物在人类中形成两种不同的复合物 细胞，但它们的功能和化学计量关系在HR仍然没有表征几十年后， 的发现BCDX 2复合物（RAD 51 B/C/D/XRCC 2）和CX 3复合物（RAD 51 C和XRCC 3）还没有被发现。 以成功地纯化用于生物化学测定。基因研究进展甚微， 任何一种旁系同源物的品系都是不可行的。该提案将利用新的方法来克服这些问题 障碍，并更好地描述了RAD 51旁系同源物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RAD 51蛋白与BRCA 2或PALB 2之间的相互作用 （BRCA 2的合作伙伴和本地化者）。第二，使用独特的方法从人细胞中纯化RAD 51旁系同源物。 和创新的协议。单独的旁系同源物，以及两个旁系同源复合物，将 以确定在刺激RAD 51介导的DNA链交换中是否存在与BRCA 2的协同作用。中 补充细胞生物学方法，我将使用一个有条件的系统，系统地消耗个人 RAD 51旁系同源物和它们各自的复合物，以观察RAD 51病灶形成是否在 DNA损伤的反应。最后，我们将与纽约大学的Eli Rothenberg博士合作， 在超分辨率显微镜下测试的假设，即RAD 51 parabolic复合物招募到DSB 在人力资源部门执行特定的职能。使用这种技术，我将创建一个招聘和保留的时间轴 在其他HR蛋白的背景下在DSB上。这些时空信息将告诉我们在HR的哪个步骤 旁系同源物是活跃的，它们的空间关系在单个DSB上。在研究完成后， 培训奖学金，RAD 51旁系同源物将得到更多的理解，申请人将有 接受了广泛的培训。多学科指导团队将为申请人的研究做好准备 独立和成功的职业生涯作为一个癌症研究员。

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.