Collaborative Functions of BRCA2 and RAD51 Paralogs in Homologous recombination

项目摘要 尽管经过几年的研究，人类RAD 51旁系同源物：RAD 51 B、RAD 51 C、RAD 51 D、XRCC 2和 XRCC 3仍然是细胞活力和DNA同源定向修复（HDR）所需的神秘蛋白质 双链断裂（DSB）。已经发现RAD 51旁系同源物存在于两种蛋白质复合物中， 人细胞：RAD 51 B/RAD 51 C/RAD 51 D/XRCC 2和RAD 51 C/XRCC 3。我们的初步数据表明， RAD 51旁系同源物与BRCA 2在特定方向上相互作用，这可能对于对细胞的机械控制很重要。 RAD 51核蛋白丝。我们在这项提案中的目标是解决生物化学和遗传学问题， BRCA 2和RAD 51旁系同源物在DNA损伤反应中的关系。我们的假设是 BRCA 2和RAD 51旁系同源物在HDR的突触前或突触后阶段一起工作，以增强 RAD 51核蛋白丝的稳定性或刺激链的侵袭性及同源性的搜索。我们将绘制 BRCA 2和RAD 51旁系同源物之间相互作用的位点。我们将确定相互作用是否 BRCA 2/RAD 51复合物对RAD 51丝的影响 动力学我们已经开发了人细胞系统，从该系统中纯化RAD 51蛋白质 单独地或以复合物（B/C/D/X2和C/X3）的形式。这些纯化的蛋白质将用于生物化学 HDR研究。BRCA 2与稳定复制叉以防止溶核降解有关 因此，我们将确定RAD 51旁系同源物是否合作， 与BRCA 2通过分析DNA纤维和超分辨率在停滞的复制叉处稳定RAD 51 显微镜总之，我们计划同时使用生物化学和遗传学方法来了解这种相互作用 BRCA 2和RAD 51旁系同源物之间的关系，以及这些蛋白质中的缺陷如何导致基因组不稳定性， 癌

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.