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: 8069321
• 负责人: PHOEBE L STEWART
• 金额: $ 22.6万
• 依托单位: VANDERBILT 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 2011-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8069321
• 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。修复由于氧化损伤和暴露于电离辐射而发生的DNA损伤对于遗传稳定性和抑制肿瘤生成至关重要。 NHEJ对于淋巴细胞中的V-D-J重组也是必不可少的，该淋巴细胞产生功能自适应免疫系统。 DNA依赖性蛋白激酶催化亚基（DNA-PKC）与其他关键成分KU和Artemis一起调节NHEJ途径。 KU70/80异二聚体是第一个识别并结合DNA在双链断裂的蛋白质的蛋白质，并募集DNA-PKCS与损伤位点。与DNA-PKC的复合物中的Artemis执行了NHEJ中V-D-J重组和DNA末端处理所需的内核分解活性。这三个组成部分中的任何一个中的突变导致人类的放射敏感性和严重的免疫缺陷。缺乏有关DNA-PKC和NHEJ复合物的高分辨率结构信息，这阻止了对其关键DNA修复活性和调节的机械理解。冷冻单粒子图像重建非常适合研究DNA-PKC和大型NHEJ复合物。该提案的具体目的是确定DNA-PKCS/Artemis/DNA，DNA-PKCS/ARTEMIS，DNA-PKCS/DSDNA和DNA- PKCS/KU/DNA复合物的dna-PKCS/DNA-PKCS/ARTEMIS，DNA-PKCS/ARTEMIS，DNA-PKCS/ARTEMIS，DNA-PKCS/ARTEMIS，DNA-PKCS/ARTEMIS的具体目的（<10E）分辨率的冷冻结构。结构分析将包括对接可用的原子分辨率结构和比较模型，以及混合冷冻蛋白结构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。对这些复合物的分子几何形状的详细知识将为NHEJ的激酶激活和核酸内切酶阶段提供深入的了解，并将能够对NHEJ途径的DNA破裂修复的分子机制产生可检验的假设。最终，我们对治疗癌症和免疫缺陷疾病的治疗能力将通过对疾病状态不当调节的基本生物学过程的分子理解来增强。 公共卫生相关性：拟议的研究在生物医学上是相关的，因为有关NHEJ复合物的结构信息将有助于回答有关这些复合物如何在DNA损伤站点组装，维修和重组过程如何指导以及如何触发多个平行途径和远距离途径之间的选择的关键问题。最终，这些信息将有助于理解和治疗癌症，严重的免疫缺陷（SCID）以及对电离辐射（RS-SCID）的敏感性。