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

项目摘要 在这个建议中，我们将应用生物化学和单分子方法来理解 非同源末端连接（NHEJ），人类中主要的DNA双链断裂（DSB）修复途径 细胞在NHEJ过程中，核心因子、末端加工因子和其他辅助因子将DNA末端连接在一起 并最终将它们连接起来。虽然已知这些单个因子的生化活性 在一定程度上，这些因子是如何组装成突触复合体的，以及它们如何与突触复合体结合的， 协调各种酶活性。为此，我们将应用单分子成像技术 非洲爪蟾卵提取物直接跟踪NHEJ复合物形成和突触末端的方法， 生理修复反应期间的真实的时间。完成以下具体目标将提供 增加对NHEJ的机制理解，这将有助于治疗靶向NHEJ的努力， 在CRISPR-Cas基因编辑过程中调节修复结果。 目的1：突触复合体在NHEJ过程中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 与错误的伙伴结合。我们已经证明，成对的末端通过两个不同的突触， 国家在修复。最初，末端保持在相对不稳定的长程突触复合体中， 过渡到稳定的短程突触复合体，其中末端准备被连接。为此，我们 将决定突触复合物的分子间相互作用的独特集合， 描述这些相互作用如何在修复过程中演变。特别是，我们将阐明如何核心NHEJ因素 XLF、XRCC 4和LIG 4有助于末端突触，并决定辅助因子如何促进突触的组装。 突触复合体 目标2：末端加工因子如何进入DNA末端？ 为了最小化异常末端加工和切除，DNA末端被Ku和其他因子快速结合。在 在前一个资助期，我们表明，即使是NHEJ相关的最终处理也受到限制，直到形成 有连接能力的短距离突触复合体该调节优先于易错末端的连接 处理.在这个目标中，我们将阐明分子步骤，使末端脱保护，并允许结束 处理.此外，我们还将确定Ku在修复过程中如何在DNA末端重塑，并检查DNA末端的结构。 通过阻止这种重塑对NHEJ产生影响。接下来，我们将确定不同的加工因素 在它们变得可接近后竞争DNA末端。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细胞中CRISPR-Cas9诱导的断裂的修复保真度。

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.