Mechanisms of Human DNA Double-Strand Break Repair via Quantitative Single-Molecule Imaging

项目摘要 本申请的目标是解决我们对中枢DNA修复途径的理解中的关键空白， 人类使用新的单分子方法。在复制的哺乳动物细胞中， 修复通过两个主要途径：非同源末端连接（NHEJ）途径，这是积极的整个 细胞周期和主要在复制期间活跃的同源重组（HR）。HR突变 蛋白质与基因组不稳定性和癌症易感性相关，而NHEJ中的突变 蛋白质导致基因组不稳定性、严重联合免疫缺陷（SCID）和IR敏感性，后者 因此，NHEJ和HR因素被认为是 有希望的抑制靶点，以提高放射和化学疗法的功效。此外，相互作用 这些途径之间的相互作用对于癌症治疗中对疗法的抗性的发展具有意义。 虽然控制NHEJ和HR的机制在正常人类发育中也起着关键作用，但我们的研究表明， 目前的知识状态，关于组织和串扰之间的这些途径和他们的 与DNA损伤反应（DDR）的相关性极小。尤其是我们对身体的了解很少 修复复合物的性质以及它们如何在DNA断裂位点组装/分解和调节; 这是因为普通的生物化学、结构和细胞生物学方法的能力有限， 提供这些信息。 在这项研究中，我们将使用一系列创新的单分子技术和测定来解决这个问题。 知识差距，并通过NHEJ和单端断裂定义DSB修复的分子机制， 复制细胞我们使用单分子生物化学方法来确定NHEJ丝蛋白的作用， 并重新检查连接DDR和NHEJ修复的基本机制。我们将使用单分子定位 显微镜和活细胞成像，以绘制体内HR/NHEJ修复中间体的组织，并确定 它们的位置依赖性，并研究它们的调节和DDR因子在通路选择中的作用。我们将 定义细胞中DSB复合物的纳米级结构，并确定它们与细胞DNA的关联 损伤响应（DDR）的因素，并表征这些是如何调制在不同类型的DSB。 结合起来，拟议的研究将解决关键的悬而未决的问题，具有巨大的潜力， 推动DNA损伤研究领域的发展。

Project summary The goal of this application is to address critical gaps in our understanding of the central DNA repair pathway in humans by using new single molecule methods. In replicating mammalian cells chromosomal breaks are repaired via two main pathways: the non-homologous end-joining (NHEJ) pathway, which is active throughout the cell cycle, and homologous recombination (HR) that is mainly active during replication. Mutations in HR proteins are associated with genome instability and predisposition to cancer, whereas mutations in NHEJ proteins result in genome instability, severe combined immunodeficiency (SCID), and IR sensitivity, the latter relevant to patient hypersensitivity to therapeutic IR. Consequently, NHEJ and HR factors are recognized as promising targets for inhibition to improve the efficacy of radiation and chemotherapy. Additionally, the interplay between these pathways has implication for the development of resistance to therapy in the treatment of cancer. Although the mechanisms that control NHEJ and HR also have key roles in normal human development, our current state of knowledge regarding the organization and crosstalk between these pathways and their correlations with the DNA damage response (DDR) is minimal. We especially know very little about the physical nature of the repair complexes and how they are assembled/disassembled and regulated at DNA break sites; this is because common biochemical, structural and cell biology approaches are limited in their capacities to provide this information. In this study we will use an array of innovative single-molecule techniques and assays to address this knowledge gap and to define the molecular mechanisms of DSB repair via NHEJ and at single-ended breaks in replicating cells. We use single-molecule biochemical methods to determine the role of NHEJ filament proteins, and reexamine basic mechanisms that link DDR and NHEJ repair. We will use single-molecule localization microscopy and live cell imaging to map the organization of HR/NHEJ repair intermediates in-vivo and determine their positional dependence, and study their regulation and the role of DDR factors in pathway choice. We will define the nanoscale architecture of DSB complexes in cells and determine their association with cellular DNA damage response (DDR) factors and characterize how these are modulated in different types of DSBs. Combined, the proposed study will address critical unanswered questions with have enormous potential for advancing the field of DNA damage research.