Collaborative Functions of BRCA2 and RAD51 Paralogs in Homologous recombination

BRCA2 和 RAD51 旁系同源物在同源重组中的协同功能

基本信息

批准号：
10431337
负责人：
Ryan Brown Jensen
金额：
$ 25.13万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-11 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10431337
关键词：
Acute Address BRCA2 gene Behavior Biochemical Biochemical Genetics Biochemistry Biological Assay Cell Line Cell Survival Cell model Cells Cellular Stress Clinical Collaborations Comb animal structure Complex Coupled DNA DNA Damage DNA Double Strand Break DNA Repair DNA analysis DNA replication fork Data Defect Diagnosis Engineering Eye Fiber Filament Future Genomic Instability Germ-Line Mutation Goals Growth Health Hereditary Malignant Neoplasm Human Human Genome Individual Investigation Kinetics Lead Link Malignant Neoplasms Maps Methods Microscopy Missense Mutation Mission Modeling Mutation Nucleoproteins Ovarian Pathogenicity Pathologic Pathway interactions Patients Phase Positioning Attribute Process Property Proteins Public Health RAD51C gene Radiation therapy Reaction Research Resolution Role Selection for Treatments Set protein Site Somatic Mutation Synapses System Testing Translations United States National Institutes of Health Variant Work XRCC2 gene XRCC3 gene base cancer cell cancer risk design experimental study gene repair genetic approach homologous recombination improved in vitro activity inhibitor insight mammary epithelium novel paralogous gene prevent protein complex protein function reconstitution repair function repaired replication stress response single molecule therapy resistant tool treatment strategy tumor

项目摘要

PROJECT SUMMARY Despite several years of investigation, the human RAD51 paralogs: RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3 remain enigmatic proteins required for cell viability and homology-directed repair (HDR) of DNA double-strand breaks (DSBs). The RAD51 paralogs have been found to exist in two protein complexes within human cells: RAD51B/RAD51C/RAD51D/XRCC2 and RAD51C/XRCC3. Our preliminary data indicate that the RAD51 paralogs interact with BRCA2 in a specific orientation likely important for mechanistic control over the RAD51 nucleoprotein filament. Our objective in this proposal is to address the biochemical and genetic relationship between BRCA2 and the RAD51 paralogs in response to DNA damage. Our hypothesis is that BRCA2 and the RAD51 paralogs work together in the pre- or post-synaptic phase of HDR to either enhance RAD51 nucleoprotein filament stability or to stimulate strand invasion and the homology search. We will map the sites of interaction between BRCA2 and the RAD51 paralogs. We will determine whether interactions are regulated by DNA damage and what impact the BRCA2/RAD51 paralog complex has on RAD51 filament dynamics. We have developed human cell systems from which to purify the RAD51 paralog proteins individually or in complexes (B/C/D/X2 and C/X3). These purified proteins will then be used for biochemical studies of HDR. BRCA2 has been linked to stabilization of replication forks to prevent nucleolytic degradation under conditions of cellular stress, and therefore, we will determine whether the RAD51 paralogs cooperate with BRCA2 to stabilize RAD51 at stalled replication forks through analyses of DNA fibers and super-resolution microscopy. In summary, we plan to use both biochemical and genetic approaches to understand the interplay between BRCA2 and the RAD51 paralogs and how defects in these proteins lead to genomic instability and cancer.

项目摘要尽管进行了数年的调查，但人类rad51旁系同源物：rad51b，rad51c，rad51d，xrcc2和 XRCC3仍然是细胞活力和同源指导修复（HDR）DNA所需的神秘蛋白双链断裂（DSB）。已经发现Rad51旁系同源物存在于两个蛋白质复合物中人类细胞：RAD51B/RAD51C/RAD51D/XRCC2和RAD51C/XRCC3。我们的初步数据表明 RAD51旁系同子在特定方向上与BRCA2相互作用，对于对机械控制很重要 RAD51核蛋白丝。我们在此提案中的目标是解决生化和遗传 BRCA2与Rad51旁系同源物之间的关系响应DNA损伤。我们的假设是 BRCA2和RAD51旁系同源物在HDR的突触前或突触后相处起作用，以增强 RAD51核蛋白细丝稳定性或刺激链入侵和同源性搜索。我们将映射 BRCA2和RAD51旁系同源物之间的相互作用位点。我们将确定互动是否是受DNA损伤调节以及BRCA2/RAD51旁系同源络合物对Rad51细丝的影响动力学。我们已经开发了纯化Rad51旁系同源蛋白的人类细胞系统单独或复合体（B/C/D/X2和C/X3）。然后，这些纯化的蛋白质将用于生化 HDR的研究。 BRCA2已与复制叉的稳定相关，以防止核酸化降解在细胞应激的条件下，因此，我们将确定RAD51旁系同源物是否合作用BRCA2通过分析DNA纤维和超分辨率在停滞的复制叉处稳定RAD51 显微镜。总而言之，我们计划使用生化和遗传方法来理解相互作用在BRCA2和RAD51旁系同源物以及这些蛋白质缺陷之间如何导致基因组不稳定性和癌症。