Defining RAD51 paralog complexes to inform their roles in genome maintenance

 描述（由申请方提供）：同源重组修复（HRR）对于准确修复可能因暴露于环境毒素、电离辐射或内源性代谢而形成的链间交联（ICL）和双链DNA断裂（DSB）至关重要。在人类中，这一过程的关键是五个RAD 51旁系同源物（RAD 51 B，RAD 51 C，RAD 51 D，XRCC2和XRCC3），它们通过相互作用，RAD 51和DNA发挥作用。然而，对RAD 51蛋白复合物的生物学功能知之甚少，尽管多年的研究，关于它们的组装和活性的机制信息一直是谜。基于突破性的初步数据，其中包括强大的可溶性表达的RAD 51 parthalamic复合物和一个RAD 51 C同系物的晶体结构，我提出了两个目标，联合收割机结合小角X射线散射和大分子晶体学，以建立准确的RAD 51 parthalamic亚复合物。这些结构模型将使用酵母双杂交和蛋白质下拉测定进行实验验证。该提案的结果，包括用于生产可溶性RAD51旁系同源物的稳健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测定铺平道路，以剖析RAD51旁系同源物在HRR和其他基因组维护途径中的功能。这项结构工作还将提供一个分子框架，从中了解临床上出现的癌症易感性RAD51突变。此外，由于RAD51旁系同源缺陷细胞对电离辐射和ICL试剂的敏感性，这项工作可以提供一个分子框架来设计特异性RAD51旁系同源抑制剂，这些抑制剂可以用于与靶向癌症治疗剂组合的合成致死性的先进疗法。总的来说，这项提议的结果将提供重要的试剂和信息结构，这将导致对RAD 51旁腺细胞功能的更好理解，并提供一个框架来理解和分类来自临床的RAD 51旁腺突变。

 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.