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

项目总结 这个应用程序的目标是解决我们对中枢DNA修复途径的理解中的关键差距 人类通过使用新的单分子方法。在复制哺乳动物细胞时，染色体断裂是 修复通过两条主要途径：非同源末端连接(NHEJ)途径，这是始终活跃的 细胞周期，以及复制过程中主要活跃的同源重组(HR)。人力资源中的突变 蛋白质与基因组不稳定和癌症易感性有关，而NHEJ基因突变 蛋白质导致基因组不稳定、严重的联合免疫缺陷(SCID)和IR敏感性，后者 与患者对治疗性IR的过敏有关。因此，NHEJ和HR因素被认为是 有望成为提高放疗和化疗疗效的抑制靶点。此外，这种相互作用 这些途径之间的相互作用对于癌症治疗中抵抗治疗的发展具有重要意义。 尽管控制NHEJ和HR的机制在正常的人类发育中也起着关键作用，但我们的 关于这些通路和它们之间的组织和串扰的当前知识状态 与DNA损伤反应(DDR)的相关性很小。我们特别是对身体的了解很少 修复复合体的性质以及它们如何在DNA断裂位点组装/分解和调节； 这是因为常见的生化、结构和细胞生物学方法在能力上受到限制。 提供此信息。 在这项研究中，我们将使用一系列创新的单分子技术和分析来解决这个问题 知识差距和确定DSB修复通过NHEJ和在单端断裂的分子机制 复制细胞。我们使用单分子生化方法来确定NHEJ丝状蛋白的作用， 并重新审查将DDR和NHEJ修复联系起来的基本机制。我们将使用单分子定位 显微镜和活细胞成像绘制体内HR/NHEJ修复中间体的组织图并确定 它们的位置依赖性，并研究它们的调节和DDR因子在路径选择中的作用。我们会 确定细胞中DSB复合体的纳米尺度结构，并确定它们与细胞DNA的结合 损害反应(DDR)因素，并表征这些因素是如何在不同类型的DSB中调制的。 综合起来，拟议的研究将解决关键的悬而未决的问题，并具有巨大的潜力 推进DNA损伤研究领域。

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.