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）修复途径解决了在细胞期间出现的DNA病变 代谢或细胞损伤的副产品。人类DSB修复途径分为两个不同的类别： 重新加入DSB分子的结束（EJ）途径，以及使用的同源性修复（HDR）途径 修复DSB分子的模板分子。细胞用来在EJ和HDR修复之间做出决定的因素 途径仍然不完全定义。许多研究表明，细胞周期调节了DSB途径 选择，然而，在细胞周期中，有利于HDR的培养物仍使用EJ修复大多数DSB。 该研究在我的实验室中的长期目标是全面定义偏向DSB的因素，以充分维修 我们可以根据单元内的初始条件来预测DSB修复结果的细节。追求这个目标 将提高我们对DNA修复和相关过程的理解，使新一代基因编辑 具有大大提高疗效的试剂，并提出了诊断和治疗人DNA修复的新策略 病理，包括癌症和衰老。 在接下来的五年中，我们将为DSB维修开发一个整体模型，该模型描述DNA修复事件 发生在DSB和模板分子上。我们生成此模型的目标是定义不可逆的 承诺在EJ/HDR之间，并了解细胞是否感觉到他们在HDR之前的能力 通过承诺。这些是细胞的重要挑战，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衡量的DSB修复能力 并行的复合物，DSB修复复合物的平行成熟在EJ/HDR中都起作用 承诺和作为这些维修途径的检查站。 EJ和HDR复合物的平行发展 在DSB分子上或在DSB和模板分子之间分裂，可以同时允许细胞 在承诺一个或另一个之前，要开发不同类型的维修。产生成熟维修的能力 承诺之前的复合物将使DNA维修的风险大大降低。我们实用的方法是 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 空前的时间和空间分辨率。我们将使用这些技术来定义蛋白质复合物如何 随着时间的推移与染色质结合，至关重要的是，DNA与DSB修复蛋白结合的链度。 测量后一个参数将使我们能够确定何时发生与EJ/HDR决策有关的事件 因此了解该决定的何时以及如何做出。我们还探索交流机制 在多个DSB修复复合物之间并行组装到染色质上。尤其是平行事件 信息丰富，因为它们表明了一个动态系统，其中细胞同时探索多个DSB修复 途径，从而保留选择，直到维修几乎完成为止。例如，模板上的事件 分子可以充当DSB分子事件的检查点，反之亦然。 这项工作将使我们能够利用我们对DSB维修的理解来影响基因编辑的新工具 结果并改善治疗工作流程。我们还预计我们的工作将开放新的询问领域， 例如，定义DSB修复复合物如何组装在一起相互作用，并与整个单元的信号传导相互作用 机制。