Determining the effects of RAD51C ovarian cancer variants

项目摘要/摘要 大约50%的高级别浆液性卵巢癌(HGSOC)缺乏同源重组 (HR)。HR是一种高保真的DNA双链断裂修复途径，它使用同源模板 精确的DNA修复。这一过程的中心蛋白质RAD51受到严格调控。一组蛋白质 调节RAD51的工作是RAD51对偶，包括RAD51C和RAD51D。总而言之，RAD51C 在8%的HR缺陷型家族性卵巢癌中，RAD51D发生突变。尽管RAD51 Paralog是 20多年前发现了它们在HR中的确切功能，以及这些蛋白的突变是如何导致HGSOC的 仍然是未知的。我们已经确定了两种可能的RAD51C蛋白亚型，它们可能分别执行 在人力资源部门的职能。RAD51C变种可以阻止任何一种亚型的蛋白质产生，已经在 32个不同的个体。因此，我们提出了对这些破坏的异构体和突变的功能分析 他们的作品。此外，我们还发现了RAD51C的突变，这种突变会破坏RAD51C与 RAD51D。我们的初步数据已经确定了RAD51C和RAD51C之间的酵母三杂交相互作用 RAD51D是人类细胞HR效率的良好指标。然而，这种RAD51C和RAD51D的相互作用 对人力资源的贡献仍不得而知。我们将研究这些变异对RAD51蛋白调节的影响 以及使用单分子荧光显微镜(C-TRAP)的细丝动力学。通过描述活动的特征 在这些RAD51C变体中，我们将揭示WT-RAD51C的功能以及RAD51C如何功能失调 突变导致HGSOC的HR不足。

Project Summary/ Abstract Approximately 50% of high grade serous ovarian cancers (HGSOC) are deficient in homologous recombination (HR). HR is a high-fidelity DNA double-strand break repair pathway which uses a homologous template for accurate DNA repair. The central protein of this process, RAD51, is tightly regulated. One group of proteins that work to regulate RAD51 are the RAD51 paralogs, including RAD51C and RAD51D. Collectively, RAD51C and RAD51D are mutated in 8% of HR deficient familial ovarian cancers. Although the RAD51 paralogs were discovered 20+ years ago their exact function in HR, and how mutation in these proteins contribute to HGSOC is still unknown. We have identified two possible RAD51C protein isoforms which may perform separate function in HR. RAD51C variants that would prevent protein production of either isoform have been identified in 32 separate individuals. Thus, we propose a functional analysis of these isoforms and mutations which disrupt their production. In addition, we have also identified mutations in RAD51C which disrupt its interaction with RAD51D. Our preliminary data has determined that the yeast-three-hybrid interaction between RAD51C and RAD51D is a good indicator of HR efficiency in human cells. Yet, how this RAD51C-RAD51D interaction contributes to HR is still unknown. We will examine the effects of these variants on RAD51 protein regulation and filament dynamics using single-molecule fluorescence microscopy (C-trap). By characterizing the activity of these RAD51C variants we will uncover both the function of WT-RAD51C and how dysfunctional RAD51C variants contribute to HR deficiency in HGSOC.