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。修复由氧化损伤和电离辐射暴露引起的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重组也是必不可少的，其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）与其他关键组分Ku和Artemis一起调节NHEJ途径的修复沿着。Ku 70/80异二聚体是第一个识别并结合DNA双链断裂末端的蛋白质，并将DNA-PKcs募集到损伤位点。与DNA-PKcs复合的Artemis执行V-D-J重组的发夹打开步骤和NHEJ中的DNA末端加工所必需的核酸内切活性。这三种成分中的任何一种突变都会导致人类的放射敏感性和严重的联合免疫缺陷。缺乏高分辨率的结构信息的DNA-PKcs和NHEJ复合物，阻碍了其关键的DNA修复活性和调控的机制的理解。CryoEM单粒子图像重建非常适合于研究DNA-PKcs和大的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（<10 E）分辨率cryoEM结构，以及使用来自蛋白质结构预测领域的新兴工具对这些NHEJ复合物进行原子水平的结构分析。结构分析将包括可用原子分辨率结构和比较模型的对接，以及混合cryoEM从头蛋白质结构预测方法的应用。这些研究将对正在进行的生物化学、遗传学和X射线晶体学研究起到高度的补充作用。这些复合物的分子几何结构的详细知识将提供深入了解NHEJ的激酶活化和核酸内切酶阶段，并将使生成的DNA断裂修复的NHEJ途径的分子机制的可测试的假设。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不适当调节的潜在生物过程的分子理解来增强。 公共卫生关系：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这些复合物如何在DNA损伤位点组装，如何指导修复和重组过程以及触发多个平行途径和结果之间选择的关键问题。最终，这些信息将有助于理解和治疗癌症，严重联合免疫缺陷（SCID）和电离辐射敏感性（RS-SCID）。