CryoEM Structural Studies of DNA-PKcs and Nonhomologous End Joining Complexes

DNA-PKcs 和非同源末端连接复合物的冷冻电镜结构研究

• 批准号: 8225317
• 负责人: PHOEBE L STEWART
• 金额: $ 23.19万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-04 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8225317
• 关键词: Binding; Biochemical Genetics; Biological Process; Catalytic Domain; Complex; Computing Methodologies; Cryoelectron Microscopy; DNA; DNA Binding; DNA Damage; DNA Double Strand Break; DNA Repair; DNA-PKcs; DNA-dependent protein kinase; Data Collection; Defect; Dependence; Development; Disease; Docking; Double Strand Break Repair; Exposure to; G22P1 gene; Generations; Genetic; Goals; Homology Modeling; Human; Human Genome; Hybrids; Immune system; Immunodeficiency and Cancer; Immunologic Deficiency Syndromes; Ionizing radiation; Knowledge; Ku70 protein; Link; Lymphocyte; Macromolecular Complexes; Malignant Neoplasms; Mammalian Cell; Methods; Modeling; Molecular; Mutation; Nature; Outcome; Pathway interactions; Phase; Phosphotransferases; Play; Process; Protein-Serine-Threonine Kinases; Proteins; Publishing; Radiation Tolerance; Recruitment Activity; Regulation; Resolution; Role; SCID Mice; Severe Combined Immunodeficiency; Site; Structure; V(D)J Recombination; XRCC5 gene; artemis; cancer genetics; comparative; density; ds-DNA; endonuclease; image reconstruction; insight; oxidative damage; particle; prevent; protein structure prediction; public health relevance; recombinational repair; repaired; tool; tumorigenesis

DESCRIPTION (provided by applicant): Nonhomologous end joining (NHEJ) serves as the primary pathway for repairing DNA double-strand breaks (DSBs) in humans. Repairing DNA damage that occurs from oxidative damage and exposure to ionizing radiation is vital for genetic stability and for suppression of oncogenesis. NHEJ is also essential for V-D-J recombination in lymphocytes, which generates a functional adaptive immune system. The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) regulates repair the NHEJ pathway along with other key components Ku and Artemis. The Ku70/80 heterodimer is the first protein to recognize and bind DNA ends at double strand breaks and recruits DNA-PKcs to the damage sites. Artemis in complex with DNA-PKcs performs the endonucleolytic activity necessary for the hairpin-opening step of V-D-J recombination and DNA end processing in NHEJ. Mutations in any of these three components results in radiosensitivity and severe combined immunodeficiency in humans. The lack of high resolution structural information on DNA- PKcs and NHEJ complexes has prevented a mechanistic understanding of their critical DNA repair activity and regulation. CryoEM single particle image reconstruction is well suited for studying DNA-PKcs and large NHEJ complexes. The specific aims of this proposal are to determine subnanometer (<10E) resolution cryoEM structures of DNA-PKcs/Artemis/DNA, DNA-PKcs/Artemis, DNA-PKcs/dsDNA, and DNA- PKcs/Ku/DNA complexes, as well as perform an atomic level structural analysis of these NHEJ complexes with emerging tools from the protein structure prediction field. The structural analysis will include docking of available atomic resolution structures and comparative models, as well as application of hybrid cryoEM de novo protein structure prediction methods. These studies will be highly complementary to ongoing biochemical, genetics, and x-ray crystallographic studies. Detailed knowledge of the molecular geometry of these complexes will provide insight into the kinase activation and endonuclease phases of NHEJ and will enable generation of testable hypotheses on molecular mechanisms underlying DNA break repair by the NHEJ pathway. Ultimately our ability to therapeutically treat cancer and immunodeficiency diseases will be enhanced by a molecular understanding of the underlying biological processes that are improperly regulated in the disease state. PUBLIC HEALTH RELEVANCE: The proposed studies are biomedically relevant in that structural information on NHEJ complexes will help to answer key questions on how these complexes assemble at DNA damage sites, how the repair and recombination processes are guided, and what triggers the choice between multiple parallel pathways and outcomes. Ultimately this information will be helpful in understanding and treating cancer, severe combined immune deficiency (SCID), and sensitivity to ionizing radiation (RS-SCID).

描述（由申请人提供）：非同源末端连接（NHEJ）是修复人类 DNA 双链断裂（DSB）的主要途径。修复因氧化损伤和电离辐射暴露而发生的 DNA 损伤对于遗传稳定性和抑制肿瘤发生至关重要。 NHEJ 对于淋巴细胞中的 V-D-J 重组也至关重要，从而产生功能性适应性免疫系统。 DNA 依赖性蛋白激酶催化亚基 (DNA-PKcs) 与其他关键成分 Ku 和 Artemis 一起调节 NHEJ 通路的修复。 Ku70/80 异二聚体是第一个识别并结合双链断裂处 DNA 末端并将 DNA-PKc 募集到损伤位点的蛋白质。 Artemis 与 DNA-PKcs 复合物执行 V-D-J 重组发夹打开步骤和 NHEJ 中 DNA 末端加工所需的核酸内切活性。这三个成分中任何一个的突变都会导致人类放射敏感性和严重的联合免疫缺陷。 DNA-PKcs 和 NHEJ 复合物高分辨率结构信息的缺乏阻碍了对其关键 DNA 修复活性和调节的机械理解。 CryoEM 单粒子图像重建非常适合研究 DNA-PKcs 和大型 NHEJ 复合物。该提案的具体目标是确定 DNA-PKcs/Artemis/DNA、DNA-PKcs/Artemis、DNA-PKcs/dsDNA 和 DNA-PKcs/Ku/DNA 复合物的亚纳米 (<10E) 分辨率冷冻电镜结构使用蛋白质结构预测领域的新兴工具对这些 NHEJ 复合物进行原子级结构分析。结构分析将包括现有原子分辨率结构和比较模型的对接，以及混合冷冻电镜从头蛋白质结构预测方法的应用。这些研究将与正在进行的生化、遗传学和 X 射线晶体学研究高度互补。对这些复合物分子几何结构的详细了解将有助于深入了解 NHEJ 的激酶激活和核酸内切酶阶段，并将能够生成关于 NHEJ 途径 DNA 断裂修复的分子机制的可检验假设。最终，我们治疗癌症和免疫缺陷疾病的能力将通过对疾病状态下受到不当调节的潜在生物过程的分子理解而得到增强。 公共健康相关性：拟议的研究具有生物医学相关性，因为 NHEJ 复合物的结构信息将有助于回答以下关键问题：这些复合物如何在 DNA 损伤位点组装、如何引导修复和重组过程以及是什么触发了多个并行之间的选择途径和结果。最终，这些信息将有助于了解和治疗癌症、严重联合免疫缺陷 (SCID) 和电离辐射敏感性 (RS-SCID)。