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: 8541597
• 负责人: PHOEBE L STEWART
• 金额: $ 21.8万
• 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/8541597
• 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（DSBs）的主要途径。修复由氧化损伤和电离辐射引起的DNA损伤对遗传稳定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，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异源二聚体是第一个识别和结合DNA双链断裂末端并将DNA- pkcs招募到损伤位点的蛋白质。Artemis与DNA- pkcs复合物在NHEJ中进行V-D-J重组和DNA末端加工的发夹打开步骤所必需的核内溶解活性。这三种成分中的任何一种发生突变都会导致人类的放射敏感性和严重的联合免疫缺陷。DNA- PKcs和NHEJ复合物的高分辨率结构信息的缺乏阻碍了对其关键DNA修复活性和调控的机制理解。CryoEM单粒子图像重建非常适合研究DNA-PKcs和大的NHEJ复合物。本课题的具体目标是确定DNA-PKcs/Artemis/DNA、DNA-PKcs/Artemis、DNA-PKcs/dsDNA和DNA-PKcs/ Ku/DNA复合物的亚纳米（<10E）分辨率低温电镜结构，并利用蛋白质结构预测领域的新兴工具对这些NHEJ复合物进行原子水平的结构分析。结构分析将包括对接现有的原子分辨率结构和比较模型，以及应用混合冷冻显微镜从头开始的蛋白质结构预测方法。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补充作用。这些复合物的分子几何结构的详细知识将提供对NHEJ的激酶激活和内切酶阶段的深入了解，并将使通过NHEJ途径对DNA断裂修复的分子机制产生可测试的假设。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中不适当调节的潜在生物过程的分子理解而得到增强。