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

可视化 DNA 断裂修复：非同源末端连接的单分子研究

基本信息

批准号：
10398909
负责人：
Joseph J. Loparo
金额：
$ 34.92万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-01 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10398909
关键词：
Address Back Binding Binding Sites Biochemical Biological Models C-terminal CRISPR/Cas technology Cell-Free System Cells Chromosomal translocation Chromosome Pairing Clustered Regularly Interspaced Short Palindromic Repeats Complex DNA DNA Binding DNA Damage DNA Double Strand Break DNA-PKcs Data Diffuse Disease Distal Double Strand Break Repair Enzymes Excision Failure Funding G22P1 gene Genes Human Individual LIG4 gene Lead Ligation Malignant Neoplasms Microscopy Molecular Mutagenesis Mutation Nonhomologous DNA End Joining Outcome Pathway interactions Phosphotransferases Physiological Polymerase Protein Kinase Proteins Proteome Reaction Regulation Role Source Synapses Tail Time Work XRCC4 gene XRCC5 gene Xenopus cancer therapy disease-causing mutation egg imaging approach insight intermolecular interaction molecular imaging nuclease prevent recruit repaired single molecule therapeutic target tumorigenesis unpublished works

项目摘要

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.

项目概要在本提案中，我们将应用生化和单分子方法来了解非同源末端连接（NHEJ），人类主要的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。虽然已知这些个体因素的生化活性各不相同在某种程度上，人们对这些因素如何组装成突触复合体以及它们如何发挥作用仍然知之甚少。各种酶的活动是协调的。为此，我们将应用单分子成像爪蟾卵提取物中的方法直接跟踪 NHEJ 复合体的形成和末端突触生理修复反应期间的实时。完成以下具体目标将提供增加对 NHEJ 机制的了解，这将有助于以 NHEJ 为治疗目标并在 CRISPR-Cas 基因编辑过程中调节修复结果。目标 1：NHEJ 期间突触复合体如何组装和进化？ DSB 形成后，DNA 末端快速突触以防止末端扩散至关重要分开并与错误的伙伴结合。我们已经证明，成对的末端穿过两个不同的突触修复期间的状态。最初末端被保持在相对不稳定的长程突触复合体中，然后转变为稳定的短程突触复合体，其中末端准备连接。为了这个目标我们将确定表征突触复合体的独特分子间相互作用集描述这些相互作用在修复过程中如何演变。特别是，我们将阐明核心 NHEJ 因素如何 XLF、XRCC4 和 LIG4 有助于末端突触并确定辅助因子如何促进突触的组装突触复合体。目标 2：末端加工因子如何接触 DNA 末端？为了最大限度地减少异常末端加工和切除，DNA 末端通过 Ku 和其他因素快速结合。在在之前的资助期间，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程突触复合体。该规定优先考虑连接而不是容易出错的末端加工。为此，我们将阐明实现末端脱保护并允许末端终止的分子步骤。加工。此外，我们将确定修复期间 Ku 在 DNA 末端如何重塑，并检查通过阻止这种重塑对 NHEJ 产生影响。接下来，我们将确定不同的处理因素如何当 DNA 末端变得可接近时，它们会竞争它们。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。