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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：
10608155
负责人：
Ryan Brown Jensen
金额：
$ 20.94万
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 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10608155
关键词：
Acute Address BRCA2 gene Behavior Biochemical Biochemistry Biological Assay Cell Line Cell Survival Cell model Cells Cellular Stress Clinical Collaborations Complex Coupled DNA DNA Damage DNA Double Strand Break DNA Repair DNA analysis DNA replication fork Data Defect Diagnosis Engineering Fiber Filament Future Genetic Genomic Instability Germ-Line Mutation Goals Growth Health Hereditary Malignant Neoplasm Human Human Genome Individual Invaded Investigation Kinetics Lead Link Malignant Neoplasms Maps Methods Missense Mutation Mission Modeling Mutation Nucleoproteins Ovarian Pathogenicity Pathologic Pathway interactions Patients Phase Poly(ADP-ribose) Polymerase Inhibitor Positioning Attribute Process Property Proteins Public Health RAD51C gene Radiation therapy Reaction Research Role Selection for Treatments Set protein Single-Stranded DNA Site Somatic Mutation System Testing Therapeutic Translations United States National Institutes of Health Variant Work XRCC2 gene XRCC3 gene cancer cell cancer risk design experimental study gene repair genetic approach homologous recombination improved in vitro activity insight mammary epithelium novel paralogous gene postsynaptic presynaptic prevent protein complex protein function protein purification reconstitution repair function repaired replication stress response single molecule superresolution microscopy 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.

项目总结尽管经过几年的研究，人类RAD51同源基因：RAD51B、RAD51C、RAD51D、XRCC2和 XRCC3仍然是细胞存活和DNA同源定向修复(HDR)所需的神秘蛋白质双链断裂(DSB)。已发现RAD51类似物存在于以下两种蛋白质复合体中人细胞：RAD51B/RAD51C/RAD51D/XRCC2和RAD51C/XRCC3。我们的初步数据显示 RAD51 Paralog与BRCA2以特定的方向相互作用，这对机械控制可能很重要 RAD51核蛋白细丝。我们在这项提案中的目标是解决生化和遗传问题 BRCA2与RAD51对DNA损伤反应的关系我们的假设是 BRCA2和RAD51类似物在HDR的突触前或突触后阶段共同作用，以增强 RAD51核蛋白长丝稳定性或刺激链侵袭及同源性搜索。我们将绘制地图 BRCA2与RAD51的相互作用位点。我们将确定交互是否受DNA损伤的调控以及BRCA2/RAD51准对数复合体对RAD51丝的影响动力学。我们已经开发了用于纯化RAD51准蛋白的人类细胞系统单独或在复合体中(B/C/D/X2和C/X3)。这些纯化的蛋白质随后将用于生化 HDR的研究。BRCA2与复制分叉的稳定有关，以防止核溶解降解在细胞应激的条件下，因此，我们将确定RAD51对偶是否合作使用BRCA2通过DNA纤维和超分辨分析将RAD51稳定在停滞的复制叉处显微镜。总而言之，我们计划同时使用生化和遗传方法来了解这种相互作用 BRCA2和RAD51之间的关系，以及这些蛋白的缺陷是如何导致基因组不稳定和癌症。