Single-Molecule Studies of Non-Homologous DNA End Joining

 描述(由申请人提供)：本申请的目标是利用新的单分子方法解决我们对非同源末端连接(NHEJ)DNA修复途径理解中的关键差距。NHEJ是DNA双链断裂(DSB)修复的主要途径，DSB是由电离辐射(IR)和化疗药物引起的最具细胞毒性的DNA损伤形式。NHEJ蛋白的突变与基因组不稳定、IR敏感性和严重的联合免疫缺陷(SCID)有关。因此，靶向抑制NHEJ通路对于IR治疗中肿瘤细胞的增敏具有重要意义。控制NHEJ的机制在癌症治疗的发展和反应中起着关键作用，但目前对NHEJ修复过程的了解有限。我们尤其对NHEJ复合体的物理性质以及它是如何组装的知之甚少，因为常见的生化、结构和细胞生物学方法在解析这些信息方面的能力有限。如果没有这一水平的了解，对导致IR敏感性和免疫缺陷的NHEJ突变以及在肿瘤细胞中抑制NHEJ的策略的洞察仍然停滞不前。在这项应用中，我们利用创新的单分子方法来定义NHEJ修复过程。我们使用一系列新的单分子生化方法来定义NHEJ的两个关键步骤：NHEJ复合体在DNA末端的组装(AIM-1)和DNA末端的突触(AIM-2)。在这些目标中，我们将确定NHEJ复杂成分的特定功能组织，它们对DNA末端化学的依赖，以及它们的临床和结构突变的后果。在AIM-3中，我们在细胞中研究NHEJ。我们将定义细胞中NHEJ复合体的纳米级结构，确定它们与细胞DNA损伤反应(DDR)因子的相关性，并表征这些因子在不同类型的DSB病变中是如何调制的。最后，我们将确定NHEJ蛋白的临床和结构突变的后果，以及NHEJ抑制剂对细胞内NHEJ复合体组织的影响。我们的结果将为利用尖端的单分子技术解决新的假说提供一个平台，具有推动DNA损伤研究领域的巨大潜力。

 DESCRIPTION (provided by applicant): The goal of this application is to address critical gaps in our understanding of the Non-homologous end joining (NHEJ) DNA repair pathway using new single molecule methods. NHEJ is the main pathway for repair of DNA double strand breaks (DSBs), the most cytotoxic form of DNA damage, resulting from Ionizing Radiation (IR) and chemotherapeutics. Mutations in NHEJ proteins are associated with genomic instability, IR sensitivity and severe combined immunodeficiency (SCID). Consequently, the targeted inhibition of the NHEJ pathway is of importance for sensitization of cancer cells in IR therapy. The mechanisms that control NHEJ play a key role in development and in response to cancer therapy, but the current state of knowledge regarding the NHEJ repair process is limited. We especially know very little about the physical nature of the NHEJ complex and how it is assembled, as common biochemical, structural and cell biology methods are limited in their capacities to resolve this information. Without this level of understanding, insights into NHEJ mutations that cause IR sensitivity and immunodeficiency and strategies for inhibiting NHEJ in neoplastic cells remain stagnant. In this application we utilize innovative single-molecule methods to define the NHEJ repair process. We use an array of new single-molecule biochemical methods to define the two critical steps of NHEJ: Assembly of NHEJ complex on DNA ends (Aim-1) and synapsis of DNA ends (Aim-2). Within these aims we will determine the specific functional organization of NHEJ complex components, their dependence on DNA end chemistry, and the consequence of their clinical and structural mutations. In Aim-3 we study NHEJ in cells. We will define the nanoscale architecture of NHEJ complexes in cells and determine their association with cellular DNA damage response (DDR) factors and characterize how these are modulated in different types of DSB lesions. Finally, we will determine the consequence of clinical and structural mutations in NHEJ proteins and the effect of NHEJ inhibitors on the organization of NHEJ complexes in cells. Our results will provide a platform for addressing novel hypotheses with cutting-edge single-molecule technology, with enormous potential for advancing the field of DNA damage research.