Defining RAD51 paralog complexes to inform their roles in genome maintenance

 描述(申请人提供)：同源重组修复(HRR)对于准确修复因暴露于环境毒素、电离辐射或内源性代谢而形成的链间交联链(ICL)和双链DNA断裂(DSB)至关重要。在人类的这一过程中，必不可少的是五个RAD51类似物(RAD51B、RAD51C、RAD51D、XRCC2和XRCC3)，它们通过彼此、RAD51和DNA的相互作用来发挥作用。然而，人们对RAD51类复合体的生物学功能知之甚少，尽管经过多年的研究，但关于它们的组装和活性的机制信息一直是个谜。基于突破性的初步数据，包括RAD51准分子络合物的稳健可溶性表达和RAD51C同源晶体结构，我提出了将小角X射线散射和大分子结晶学相结合来构建准确的RAD51准分子络合物的两个目标。这些结构模型将使用酵母双杂交和蛋白质下拉试验进行实验验证。这一建议的结果，包括生产可溶性RAD51并列基因的可靠方法和设计功能分离突变的分子基础，将为我和该领域的其他人进行新的生化和生物学分析铺平道路，以剖析RAD51并列基因在HRR和其他基因组维持途径中的功能。这项结构性工作还将提供一个分子框架，用于了解临床上出现的易患癌症的RAD51平行突变。此外，由于RAD51 Paralog缺陷细胞对电离辐射和ICL试剂的敏感性，这项工作可能提供一个分子框架来设计特定的RAD51 Paralog抑制剂，该抑制剂可能对通过合成致命性与靶向癌症治疗相结合的先进疗法有用。总之，这项建议的结果将提供重要的试剂和信息结构，将有助于更好地了解RAD51 Paralog细胞功能，并提供一个框架，以了解和分类来自临床的RAD51 Paralog突变。

 DESCRIPTION (provided by applicant): Homologous recombinational repair (HRR) is essential for the accurate repair of inter-strand crosslinks (ICLs) and double stranded DNA breaks (DSBs) that can form from exposure to environmental toxins, ionizing radiation or endogenous metabolism. Essential to this process in humans are five RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3), which carry out their roles through interaction with one another, RAD51 and DNA. However, the biological function of RAD51 paralog complexes are poorly understood, and despite years of study mechanistic information about their assemblies and activities has been enigmatic. Based on breakthrough preliminary data that includes robust soluble expression of RAD51 paralog complexes and a RAD51C homolog crystal structure, I propose two aims that combine small-angle X-ray scattering and macromolecular crystallography to build accurate RAD51 paralog subcomplexes. These structural models will be experimentally validated using yeast two-hybrid and protein pull-down assays. Results from this proposal, including robust methods for the production of soluble RAD51 paralogs and a molecular basis from which to design separation-of-function mutations, will pave the way for new biochemical and biological assays by myself and others in the field to dissect the function of RAD51 paralogs in HRR and other genome maintenance pathways. This structural work will also provide a molecular framework from which to understand cancer predisposing RAD51 paralog mutations that are emerging from the clinic. Furthermore, due to the sensitivity of RAD51 paralog-deficient cells to ionizing radiation and ICL agents, this work may provide a molecular framework to design specific RAD51 paralog inhibitors that could be useful for advanced therapies that work by synthetic lethality in combination with targeted cancer therapeutics. Collectively, results from this proposal will provide important reagents and informative structures that will lead to greater understanding of RAD51 paralog cellular functions, and provide a framework to understand and classify RAD51 paralog mutations from the clinic.