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

DNA双链断裂(DSB)修复途径解决细胞内DNA损伤 新陈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： 重新加入DSB分子的末端连接(EJ)通路，以及使用 用于修复DSB分子的模板分子。细胞用来决定EJ和HDR修复的因素 路径仍然没有完全确定。许多研究表明，细胞周期调节DSB途径 选择，然而，在细胞周期中有利于HDR的点被阻止的培养仍然使用EJ修复大多数DSB。 我实验室研究的长期目标是全面定义影响DSB修复的因素 我们可以根据细胞内的初始条件预测DSB修复结果的详细信息。追求这一目标 将提高我们对DNA修复和相关过程的理解，使新一代基因编辑成为可能 大大提高疗效的试剂，并提出诊断和治疗人类DNA修复的新策略 病理，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修复的整体模型 发生在DSB和模板分子上。我们在生成此模型时的目标是定义不可逆转的 在EJ/HDR和了解细胞是否感觉到它们的能力之前执行HDR的承诺步骤 传递承诺。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 或者基因组的不稳定性。我们假设细胞具有迄今未被测量的发展DSB修复的能力 DSB修复复合体的平行成熟在EJ/HDR中都起着作用 并作为这些修复道路的检查站。EJ和HDR复合体的并行开发 无论是在DSB分子上还是在DSB和模板分子之间分裂都会使细胞同时 在致力于一种或另一种之前，开发不同类型的修复。产生成熟修复的能力 承诺之前的复合体将使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 前所未有的时间和空间分辨率。我们将使用这些技术来定义蛋白质复合体是如何 随着时间的推移，与染色质有关，关键的是，DNA与DSB修复蛋白结合的链状结构。 测量后一个参数将允许我们确定与EJ/HDR决定有关的事件发生的时间 从而理解这一决定是在什么时候以及如何做出的。我们还探索了沟通的机制 在平行组装到染色质上的多个DSB修复复合体之间。平行活动特别是 提供信息，因为它们表明了一个动态系统，在该系统中，细胞同时探索多个DSB修复 路径，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能够利用我们对DSB修复的理解来影响基因编辑 结果，并改进治疗工作流程。我们也期待着我们的工作将开辟新的调查领域， 例如，定义组装的DSB修复复合体如何相互作用以及如何与细胞范围内的信号相互作用 机械装置。

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.