Structural Cell Biology of DNA Repair Machines

描述(申请人提供)：我们正在研究切除DNA损伤的DNA修复复合体的结构组织，以及突变和小分子抑制剂破坏这些蛋白质-蛋白质相互作用的功能后果。许多类型的DNA损伤都有不同的修复途径，而针对DNA损伤产生的翻译后修饰控制着修复复合体的差异组装，为调节途径选择提供了一种机制。DNA维持活动中与癌症相关的缺陷可以通过以机制为基础的抑制剂靶向剩余的修复活动来进行治疗。我们的工作主要集中在碱基切除和核苷酸切除修复途径产生的DNA单链断裂修复机制上。我们正在研究DNA损伤切除复合体在体外和培养细胞中的物理组装，以及DNA切割与末端加工和连接的耦合机制。纯化的DNA修复复合体的小角X射线散射揭示了动态构象状态，我们认为这种动态构象状态对于将DNA修复中间产物移交给路径中的后续酶是重要的。高分辨晶体结构和小分子筛选实验正被用于预测和识别修复蛋白相互作用的抑制物，这些蛋白是抗肿瘤治疗的候选药物，并在DNA损伤反应中作为细胞生理的可逆化学探针。这种综合方法利用了项目1、2和6中研究人员在分析和生物材料方面的广泛专业知识，以及SBDR计划的表达和分子生物学核心以及结构细胞生物学核心生产蛋白质和从结构上评估修复复合体的独特能力。

DESCRIPTION (provided by applicant): We are studying the structural organization of DNA repair complexes that excise DNA damage and the functional consequences of disrupting these protein-protein interactions by mutation and small molecule inhibitors. Alternative pathways of repair are available for many types of DNA damage, and posttranslational modifications generated in response to DNA damage control the differential assembly of repair complexes, providing a mechanism for regulating pathway choice. Cancer-associated defects in DNA maintenance activities can be exploited therapeutically by targeting the remaining repair activities with mechanism-based inhibitors. Our work focuses on the mechanisms of repairing DNA single strand breaks generated by the base excision and nucleotide excision repair pathways. We are studying the physical assembly of DNA damage excision complexes in vitro and in cultured cells, and the mechanism of coupling DNA cleavage to end processing and ligation. Small angle x-ray scattering of purified DNA repair complexes reveals dynamic conformational states that we propose are important for handoffs of DNA repair intermediates to successive enzymes in a pathway. High resolution crystal structures and small molecule screening experiments are being used to predict and identify inhibitors of repair protein interactions, which are candidates for anti-tumor therapies and serve as reversible chemical probes of cellular physiology during DNA damage responses. This integrated approach takes advantage of the broad expertise of investigators in Projects 1, 2, and 6 for assays and biological materials, as well as the unique capabilities of the Expression and Molecular Biology Core and the Structural Cell Biology Core of the SBDR Program to produce proteins and structurally evaluate repair complexes.