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

基本信息

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
项目类别：
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&apos 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

项目摘要

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破坏的分子途径对于对抗这些途径至关重要毁灭性疾病。遗传性对癌症的敏感性可能是由乳房种系突变引起的癌症敏感性基因BRCA1和BRCA2。 BRCA基因代码涉及同源的蛋白质 DNA双链断裂（DSB）的重组（HR）修复和反向复制的保护叉子。尤其是BRCA2对HR至关重要，因为它将Rad51加载到形成A 核蛋白丝。五个RAD51旁系同源物：Rad51b，Rad51C，Rad51d，XRCC2和XRCC3是A 与Rad51同源的蛋白质家族已与人力资源调节有关，生殖线突变具有与家族性乳腺癌和卵巢癌有关。 Rad51旁系同源物在人类中形成两个不同的复合物细胞，但它们的功能和人力资源的化学计量关系在它们之后的几十年保持了几十年发现。 BCDX2复合物（RAD51B/C/D/XRCC2）和CX3复合物（RAD51C和XRCC3）尚未成功纯化用于生化测定。作为基因敲除细胞，遗传研究几乎没有取得进展任何一个旁系同源物的线都不可行。该建议将利用新颖的方法来克服这些障碍物和更好地表征了Rad51旁系同源物在人力资源中的作用。第一种方法将确定特定域是否介导RAD51旁系同源蛋白与BRCA2或PALB2之间的相互作用是否介导（BRCA2的合作伙伴和本地化）。其次，Rad51旁系同源物将使用独特的和我们制定的创新协议。单个旁系同源物以及两个旁系同源络合物将进行测试以确定BRCA2是否存在刺激RAD51介导的DNA链交换中的协同作用。在互补的细胞生物学方法，我将使用条件系统系统地耗尽个人 Rad51旁系同源物及其各自的复合物，以观察Rad51焦点形成是否在对DNA损伤的反应。最后，与纽约大学的Eli Rothenberg博士合作，我们将利用他的专业知识在超分辨率显微镜中，测试了Rad51旁系同源络合物被募集到DSB的假设在人力资源期间执行特定功能。使用此技术，我将创建一个招聘和保留时间表在其他HR蛋白的背景下DSB。此时空信息将告知我们在人力资源部的哪个步骤旁系同源物是活跃的，它们在单个DSB处的空间关系。研究完成后培训奖学金，RAD51旁系同源物将被更加了解，申请人将拥有接受了广泛的培训。多学科指导团队将为研究申请人做好准备独立性和作为癌症研究员的成功职业。