Defining DNA resection and protein localization changes that occur during DSB repair

定义 DSB 修复过程中发生的 DNA 切除和蛋白质定位变化

• 批准号: 10826403
• 负责人: Chris Richardson
• 金额: $ 7.32万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA BARBARA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-12 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10826403
• 关键词: Aging; Binding; Biological Process; Categories; Cell Cycle; Cell Death; Cell physiology; Cells; Chromatin; Communication; DNA; DNA Binding; DNA Double Strand Break; DNA Repair; DNA biosynthesis; DNA lesion; Data; Development; Diagnosis; Double Strand Break Repair; Event; Excision; FANCD2 protein; Generations; Genes; Genomic Instability; Genomics; Goals; Histones; Human; Lysine; Malignant Neoplasms; Measures; Metabolism; Modeling; Mutation; Natural Immunity; Nucleosomes; Outcome; Pathology; Pathway interactions; Positioning Attribute; Process; Proteins; Proteomics; Reagent; Repair Complex; Research; Resolution; Signal Transduction; Techniques; Testing; Therapeutic; Time; Work; cell injury; chemotherapy; dynamic system; extrachromosomal DNA; falls; human DNA; human disease; improved; mutant; parent grant; preservation; protein complex; repaired; tool

Project Summary/Abstract—R35 PARENT GRANT DNA double strand break (DSB) repair pathways resolve DNA lesions that arise during cellular metabolism or as the by-product of cell damage. Human DSB repair pathways fall into two distinct categories: end joining (EJ) pathways that rejoin the DSB molecule, and homology directed repair (HDR) pathways that use a template molecule to repair the DSB molecule. The factors that cells use to decide between EJ and HDR repair pathways remain incompletely defined. Many studies have shown that the cell cycle regulates DSB pathway choice, yet cultures arrested at points in the cell cycle that favor HDR still repair the majority of DSBs using EJ. The long-term goal of the research in my lab is to comprehensively define factors that bias DSB repair in sufficient detail that we can predict DSB repair outcomes based on the initial conditions inside a cell. Pursuit of this goal will improve our understanding of DNA repair and related processes, enable new generations of gene editing reagents with greatly increased efficacy, and suggest new strategies to diagnose and treat human DNA repair pathologies, including cancer and aging. Over the next five years, we will develop a holistic model for DSB repair that describes DNA repair events occurring on the DSB and template molecules. Our goals in generating this model are to define the irreversible commitment step between EJ/HDR and to understand if cells sense their capacity to perform HDR before they pass commitment. These are important challenges for the cell, because inappropriate HDR can cause cell death or genomic instability. We hypothesize that cells have the heretofore unmeasured ability to develop DSB repair complexes in parallel, and that parallel maturation of DSB repair complexes plays a role both in the EJ/HDR commitment and as a checkpoint for these repair pathways. Parallel development of EJ and HDR complexes either on the DSB molecule or split between the DSB and template molecule would allow cells to simultaneously develop different types of repair before committing to one or the other. The ability to generate mature repair complexes prior to commitment would make DNA repair substantially less risky. Our practical approach is to develop genomic and proteomic techniques that allow us to measure DSB repair intermediates with unprecedented temporal and spatial resolution. We will use these techniques to define how protein complexes associate with chromatin over time and, crucially, the strandedness of DNA bound to DSB repair proteins. Measuring this latter parameter will allow us to determine when events occur in relation to the EJ/HDR decision and thus understand when and how this decision is made. We also explore mechanisms of communication between multiple DSB repair complexes assembled in parallel onto chromatin. Parallel events are especially informative because they indicate a dynamic system in which cells simultaneously explore multiple DSB repair pathways, thereby preserving choice until repair is nearly complete. For example, events on the template molecule may act as a checkpoint for events on the DSB molecule, or vice versa. This work will enable new tools that leverage our understanding of DSB repair to influence gene editing outcomes and to improve therapeutic workflows. We also anticipate that our work will open new fields of inquiry, for example defining how DSB repair complexes assembled interact with each other and with cell-wide signaling mechanisms.

项目摘要/摘要—R35 家长补助金 DNA 双链断裂 (DSB) 修复途径可解决细胞过程中出现的 DNA 损伤 代谢或作为细胞损伤的副产品人类 DSB 修复途径分为两种。 不同的类别：重新连接 DSB 分子的末端连接 (EJ) 途径和同源性 使用模板分子修复 DSB 分子的定向修复 (HDR) 途径。 细胞用来决定 EJ 和 HDR 修复途径的因素仍然不完全 许多研究表明，细胞周期调节 DSB 途径的选择。 在细胞周期中有利于 HDR 的点处停止的培养物仍然可以使用以下方法修复大多数 DSB EJ. 我实验室研究的长期目标是全面定义偏见因素。 DSB 修复足够详细，我们可以根据初始 DSB 修复结果来预测 DSB 修复结果 对细胞内条件的追求将提高我们对 DNA 修复和修复的理解。 相关工艺，使新一代基因编辑试剂的性能大大提高 功效，并提出诊断和治疗人类 DNA 修复病理的新策略， 包括癌症和衰老。 在接下来的五年里，我们将开发一个描述 DNA 的 DSB 修复整体模型 DSB 和模板分子上发生的修复事件是我们生成此模型的目标。 的目的是定义 EJ/HDR 之间不可逆的承诺步骤并了解细胞是否 在做出承诺之前了解他们执行 HDR 的能力。 细胞面临的挑战，因为不适当的 HDR 会导致细胞死亡或基因组不稳定。 我们发现细胞具有迄今为止无法测量的 DSB 修复能力 DSB 修复复合物的平行成熟在以下方面均发挥作用： EJ/HDR 承诺并作为这些并行开发的检查点。 EJ 和 HDR 复合物位于 DSB 分子上或在 DSB 和模板之间分离 分子将允许细胞在进行修复之前同时进行不同类型的修复 在承诺之前产生成熟修复复合物的能力将是其中之一。 我们的实用方法是开发基因组和基因组修复技术，从而大大降低 DNA 修复的风险。 蛋白质组学技术使我们能够以前所未有的方式测量 DSB 修复中间体 我们将使用这些技术来定义蛋白质如何复合。 随着时间的推移，与染色质相关，最重要的是，与 DSB 修复结合的 DNA 链 测量后一个参数将使我们能够确定事件何时发生。 与 EJ/HDR 决策相关，从而了解我们何时以及如何做出该决策。 还探索了组装的多个 DSB 修复复合物之间的通讯机制 平行于染色质 平行事件特别有用，因为它们表明了 细胞同时探索多种 DSB 修复途径的动态系统，从而 保留选择直到修复接近完成。例如，模板分子上的事件。 可能充当 DSB 分子事件的检查点，反之亦然。 这项工作将使新工具能够利用我们对 DSB 修复的理解来影响基因 我们还预计我们的工作将编辑结果并改进治疗工作流程。 开辟新的研究领域，例如定义 DSB 修复复合物如何组装相互作用 彼此之间以及细胞范围内的信号传导机制。

项目成果

Interstrand crosslinking of homologous repair template DNA enhances gene editing in human cells.

• DOI: 10.1038/s41587-022-01654-y
• 发表时间: 2023-10
• 期刊: NATURE BIOTECHNOLOGY
• 影响因子: 46.9
• 作者: Ghasemi, Hannah I.;Bacal, Julien;Yoon, Amanda C.;Tavasoli, Katherine U.;Cruz, Carmen;Vu, Jonathan T.;Gardner, Brooke M.;Richardson, Chris D.
• 通讯作者: Richardson, Chris D.