Defining RAD51 paralog complexes to inform their roles in genome maintenance

定义 RAD51 旁系同源复合体以了解其在基因组维护中的作用

• 批准号: 8952945
• 负责人: Gareth J Williams
• 金额: $ 29.48万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-15 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8952945
• 关键词: ATP phosphohydrolase; Address; Amino Acid Substitution; BRCA1 gene; BRCA2 gene; Binding; Biochemical; Biological; Biological Assay; Biological Process; Biology; Cell physiology; Cells; Cellular Assay; Clinic; Clinical; Complex; Crystallography; DNA; DNA Double Strand Break; DNA Interstrand Crosslinking; DNA Sequence; Data; Development; Exposure to; Future; Genetic Polymorphism; Genome; Genomic Instability; Health; Homologous Gene; Human; Hydrolysis; In Vitro; Ionizing radiation; Laboratories; Lead; Link; Macromolecular Complexes; Maintenance; Malignant Neoplasms; Malignant neoplasm of ovary; Metabolism; Methods; Mining; Modeling; Molecular; Mutation; Outcome; Pathway interactions; Patients; Play; Predisposition; Process; Production; RAD51C gene; Reagent; Research; Resolution; Roentgen Rays; Role; Structural Models; Structure; System; Testing; Therapeutic; Toxic Environmental Substances; Walkers; Work; X-Ray Crystallography; XRCC2 gene; XRCC3 gene; Yeasts; base; cancer therapy; crosslink; design; disease-causing mutation; hybrid protein; inhibitor/antagonist; interest; macromolecular assembly; malignant breast neoplasm; paralogous gene; public health relevance; recombinational repair; repaired; scale up; structural biology; yeast two hybrid system

 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.

 描述（由适用提供）：同源重组修复（HRR）对于准确修复跨链接交联（ICL）和双链DNA断裂（DSB）至关重要，这些交联（ICLS）和双链DNA断裂（DSB）可以从暴露于环境毒素，电离辐射或内源性代谢中形成。对于人类中的这一过程至关重要的是五个RAD51旁系同源物（Rad51b，Rad51c，Rad51d，XRCC2和XRCC3），它们通过彼此相互作用，Rad51和DNA来扮演角色。但是，RAD51旁系同源物复合物的生物学功能知之甚少，所需的多年研究机械信息有关其组装和活动是神秘的。基于突破性的初步数据，包括RAD51旁系同源络合物的稳健固体表达和RAD51C同源晶体结构，我提出了两个将小角X射线散射和大分子晶体学结合的目标，以构建准确的RAD51 paralog paralog子复合物。这些结构模型将通过酵母两杂交和蛋白质下拉测定法对实验验证。该提案的结果，包括用于生产固体RAD51旁系同源物的可靠方法，以及从中设计功能分离突变的分子基础，将为我自己和领域中其他人的新生物化学和生物学测定铺平道路，以解剖HRR和其他基因组维持途径RAD51旁系同源物的功能。这项结构性工作还将提供一个分子框架，从诊所中出现的RAD51旁系同源物突变易感性。此外，由于RAD51旁系同型缺陷细胞对电离辐射和ICL剂的敏感性，这项工作可能为设计特定的RAD51旁系同源物抑制剂提供了一个分子框架，这些框架可用于与靶向癌症结合使用合成致死性的晚期疗法。总的来说，该提案的结果将提供重要的试剂和信息结构，从而使RAD51旁系同源物细胞功能更加了解，并提供一个框架来了解和对诊所的Rad51旁系同源物进行分类。