Visualizing DNA break repair: single-molecule studies of non-homologous end joining

项目摘要 在这项建议中，我们将应用生物化学和单分子方法来了解 非同源末端连接(NHEJ)是人类主要的DNA双链断裂(DSB)修复途径 细胞。在NHEJ期间，核心因素、末端加工因素和其他辅助因素将DNA末端系在一起 并最终将它们结扎起来。虽然已知这些个体因素的生化活动是不同的 在一定程度上，这些因素是如何组装成突触复合体的，以及它们的 各种酶的活动是协调的。为此，我们将应用单分子成像技术 非洲爪蛙卵提取液直接跟踪NHEJ复合体形成和结束突触的方法 在生理修复反应期间的实时。完成以下具体目标将提供 增加了对NHEJ的机械性理解，这将有助于在治疗上针对NHEJ和 在CRISPR-Cas基因编辑过程中调节修复结果。 目的1：NHEJ期间突触复合体是如何组装和进化的？ 在DSB形成时，DNA末端迅速突触以防止末端扩散是至关重要的 分开并加入了错误的搭档。我们已经证明，成对的一端通过两个不同的突触 维修过程中的状态。最初的末端是在相对不稳定的远程突触复合体中 过渡到稳定的短程突触复合体，两端准备结扎。为了实现这一目标，我们 将确定表征突触复合体的独特的分子间相互作用集，并 描述这些交互作用在修复过程中如何演变。特别是，我们将阐明NHEJ的核心因素是如何 XLF、XRCC4和LIG4参与末端突触，并决定辅助因子如何促进 突触复合体。 目标2：末端加工因子如何获得DNA末端？ 为了最大限度地减少异常末端加工和切除，DNA末端被Ku和其他因素迅速结合。在……里面 在上一个资助期，我们表明即使是与NHEJ相关的末端加工也受到限制，直到形成 连接能力强的短程突触复合体。这一规定优先于结扎，而不是容易出错的末端 正在处理。在这个目标中，我们将阐明实现末端去保护和允许末端的分子步骤 正在处理。此外，我们将确定修复过程中DNA末端的Ku是如何重塑的，并检查 阻止这种重塑对NHEJ的影响。接下来，我们将确定不同的处理因素 在DNA末端变得可访问后，竞争它们。最后，我们将把我们的机械洞察力应用到 调节末端加工以降低CRISPR-Cas9诱导的细胞断裂修复的保真度。

Project Summary In this proposal we will apply biochemical and single-molecule approaches to understand the mechanism of non-homologous end joining (NHEJ), the primary DNA double strand break (DSB) repair pathway in human cells. During NHEJ, core factors, end processing factors and other accessory factors tether DNA ends together and ultimately ligate them. While the biochemical activities of these individual factors are known to varying extents, it remains poorly understood how these factors assemble into a synaptic complex and how their various enzymatic activities are coordinated. To that end, we will apply single-molecule imaging approaches in Xenopus egg extract to directly follow NHEJ complex formation and end synapsis in real time during a physiological repair reaction. Completion of the specific aims below will provide an increased mechanistic understanding of NHEJ, which will aid efforts to therapeutically target NHEJ and to modulate repair outcomes during CRISPR-Cas gene editing. Aim 1: How does the synaptic complex assemble and evolve during NHEJ? Upon DSB formation it is critical that DNA ends are rapidly synapsed so as to prevent the ends from diffusing apart and joining with the wrong partner. We have shown that paired ends pass through two distinct synaptic states during repair. Initially ends are held in a relatively unstable long-range synaptic complex before transitioning to a stable short-range synaptic complex in which the ends are poised to be ligated. In this aim we will determine the unique sets of intermolecular interactions that characterize the synaptic complexes and describe how these interactions evolve during repair. In particular, we will elucidate how the core NHEJ factors XLF, XRCC4 and LIG4 contribute to end synapsis and determine how accessory factors facilitate assembly of the synaptic complexes. Aim 2: How do end processing factors gain access to DNA ends? To minimize aberrant end processing and resection, DNA ends are rapidly bound by Ku and other factors. In the prior funding period, we showed that even NHEJ-associated end processing is restricted until formation of the ligation-competent short-range synaptic complex. This regulation prioritizes ligation over error-prone end processing. In this aim we will elucidate the molecular steps that enable end deprotection and allow for end processing. Furthermore, we will determine how Ku is remodeled on DNA ends during repair and examine the consequences on NHEJ by blocking this remodeling. Next, we will determine how different processing factors compete for DNA ends after they become accessible. Finally, we will apply our mechanistic insight into the regulation of end processing to decrease the fidelity of repair of CRISPR-Cas9 induced breaks in cells.