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: 10468176
• 负责人: Chris Richardson
• 金额: $ 35.57万
• 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/10468176
• 关键词: Aging; 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; Development; Diagnosis; Double Strand Break Repair; Event; Excision; Generations; Genes; Genomic Instability; Genomics; Goals; Human; Malignant Neoplasms; Measures; Metabolism; Modeling; Natural Immunity; Outcome; Pathology; Pathway interactions; Play; Process; Proteins; Proteomics; Reagent; Repair Complex; Research; Resolution; Role; Signal Transduction; Techniques; Therapeutic; Time; Work; base; cell injury; chemotherapy; dynamic system; falls; human DNA; human disease; improved; preservation; protein complex; repaired; tool

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.

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修复途径分为两个不同的类别： 末端连接（EJ）途径，其重新连接DSB分子，以及同源定向修复（HDR）途径，其使用 一个模板分子来修复DSB分子。细胞用来决定EJ和HDR修复的因素 路径仍然不完全确定。许多研究表明细胞周期调控DSB通路 然而，在细胞周期中有利于HDR的点处停滞的培养物仍然使用EJ修复大多数DSB。 我实验室研究的长期目标是全面定义使DSB修复偏向充分的因素。 我们可以根据细胞内部的初始条件来预测DSB修复结果。实现这一目标 将提高我们对DNA修复和相关过程的理解，使新一代的基因编辑成为可能， 试剂的功效大大提高，并提出了诊断和治疗人类DNA修复的新策略 病理学，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修复事件的DSB修复整体模型。 发生在DSB和模板分子上。我们生成这个模型的目标是定义不可逆的 在EJ/HDR之间的承诺步骤，并了解细胞是否感觉到他们的能力，以执行HDR之前， 通过承诺。这些都是细胞面临的重要挑战，因为不适当的HDR会导致细胞死亡 或基因组不稳定性。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EJ/HDR中起作用， 承诺和作为这些修复途径的检查点。EJ和HDR复合物的并行开发 无论是在DSB分子上还是在DSB和模板分子之间分裂， 在承诺一种或另一种之前，开发不同类型的修复。产生成熟修复的能力 承诺前的复合物将使DNA修复的风险大大降低。我们的实际做法是 开发基因组和蛋白质组技术，使我们能够测量DSB修复中间体， 前所未有的时空分辨率。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修复蛋白结合的DNA链。 测量后一个参数将使我们能够确定何时发生与EJ/HDR决策相关的事件 从而了解何时以及如何做出这个决定。我们还探索沟通机制 在平行组装到染色质上的多个DSB修复复合物之间。平行事件尤其 因为它们指示细胞同时探索多个DSB修复的动态系统， 通路，从而保留选择，直到修复接近完成。例如，模板上的事件 分子可以充当DSB分子上事件的检查点，反之亦然。 这项工作将使新的工具能够利用我们对DSB修复的理解来影响基因编辑 结果和改善治疗工作流程。我们还期望我们的工作将开辟新的研究领域， 例如定义组装的DSB修复复合物如何彼此相互作用以及与细胞范围的信号传导相互作用 机制等