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

描述(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。修复因氧化损伤和暴露于电离辐射而造成的DNA损伤对于遗传稳定和抑制肿瘤发生至关重要。NHEJ对淋巴细胞中的V-D-J重组也是必不可少的，后者产生一个功能性的适应性免疫系统。DNA依赖蛋白激酶催化亚单位(DNA-PKcs)与其他关键成分Ku和Artemis一起调节NHEJ途径的修复。Ku70/80异源二聚体是第一个在双链断裂时识别和结合DNA末端并将DNA-PKcs招募到损伤部位的蛋白质。在NHEJ中，Artemis与DNA-PKcs形成复合体，具有V-D-J重组发夹打开步骤和DNA末端加工所必需的内切核活性。这三种成分中的任何一种突变都会导致人类对辐射敏感和严重的联合免疫缺陷。缺乏关于DNA-PKcs和NHEJ复合体的高分辨率结构信息，阻碍了对它们关键的DNA修复活性和调控的机械性理解。低温EM单粒子图像重建非常适合于DNA-PKcs和大型NHEJ复合体的研究。这项建议的具体目的是确定DNA-PKcs/Artemis/DNA、DNA-PKcs/Artemis、DNA-PKcs/dsDNA和DNA-PKcs/Ku/DNA复合体的亚纳米(&lt；10e；10e)分辨低温EM结构，并利用蛋白质结构预测领域的新兴工具对这些NHEJ复合体进行原子级结构分析。结构分析将包括对接现有的原子分辨结构和比较模型，以及应用混合低温EM从头开始蛋白质结构预测方法。这些研究将是对正在进行的生化、遗传学和X射线结晶学研究的高度补充。对这些复合体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相，并将能够产生关于NHEJ途径导致DNA断裂修复的分子机制的可验证的假说。最终，我们治疗癌症和免疫缺陷疾病的能力将通过对潜在生物过程的分子理解而得到增强，这些潜在的生物过程在疾病状态下受到不适当的调控。 公共卫生相关性：拟议的研究具有生物医学相关性，因为关于NHEJ复合体的结构信息将有助于回答这些复合体如何在DNA损伤部位组装、修复和重组过程如何指导以及是什么触发了在多个平行途径和结果之间进行选择的关键问题。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)和对电离辐射的敏感性(RS-SCID)。

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).