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.

项目概要/摘要-R35 PAGLENT GRANT DNA双链断裂（DSB）修复途径解决细胞周期中出现的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 定向修复（HDR）途径，其使用模板分子来修复DSB分子。的 细胞用来决定EJ和HDR修复途径的因素仍然不完全 定义了许多研究表明，细胞周期调节DSB途径的选择，但 在细胞周期中有利于HDR的点停滞的培养物仍然使用 EJ我实验室研究的长期目标是全面定义导致偏见的因素 足够详细的DSB修复，我们可以根据最初的DSB修复结果预测DSB修复结果。 细胞内的条件。追求这一目标将提高我们对DNA修复的理解， 相关工艺，使新一代基因编辑试剂与 有效性，并提出了诊断和治疗人类DNA修复病理学的新策略， 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修复的整体模型， DSB和模板分子上发生的修复事件。我们创建这个模型的目标 是定义EJ/HDR之间的不可逆承诺步骤，并了解细胞是否 在他们通过承诺之前感受他们执行人类发展报告的能力。这些都是重要 这对细胞来说是一个挑战，因为不适当的HDR会导致细胞死亡或基因组不稳定。 我们假设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两个方面都起作用： EJ/HDR的承诺，并作为这些修复途径的检查点。并行开发 在DSB分子上或在DSB和模板之间分裂的EJ和HDR复合物 一种分子可以让细胞在进行修复之前， 两个人之间在承诺之前产生成熟的修复复合物的能力将 使DNA修复的风险大大降低。我们的实际方法是开发基因组和 蛋白质组学技术，使我们能够测量DSB修复中间体， 时间和空间分辨率我们将使用这些技术来定义蛋白质复合物 随着时间的推移，与染色质相关，关键是，与DSB修复结合的DNA链 proteins.测量后一个参数将使我们能够确定事件何时发生， 与EJ/HDR决定的关系，从而了解何时以及如何做出这一决定。我们 还探索了组装的多个DSB修复复合物之间的通信机制 平行地附着在染色质上。平行事件特别具有信息性，因为它们表明 动态系统，其中细胞同时探索多个DSB修复途径，从而 保留选择权直到修复接近完成。例如，模板分子上的事件 可以作为DSB分子上事件的检查点，反之亦然。 这项工作将使新的工具，利用我们对DSB修复的理解，影响基因 编辑结果并改善治疗工作流程。我们还预计，我们的工作将 开辟新的研究领域，例如定义DSB修复复合物组装如何相互作用 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.