Functional interactions of the DNA double strand break response with nuclear architecture

环境因素(如阳光、自然生成的氡)和正常新陈代谢会对所有生物体造成DNA损伤。最危险的DNA损伤类型之一是双链断裂(DSB)，即双螺旋的两侧断裂。这些断裂的修复虽然通常是准确的，但可能会导致DNA序列改变、染色体异常或细胞死亡。我们的实验室研究了感知和修复DSB的分子信号通路，以防止细胞死亡或细胞基本特征的永久性变化。*最近，我们专门研究了这些DSB信号通路如何与其他细胞过程相互作用，如基因表达。为了研究这些途径的相互作用，我们设计了几个哺乳动物细胞模型，在这些模型中，我们可以在DNA的特定位置诱导DSB。这些系统虽然是有价值的型号，但在电池中可以生产DSB的地点数量有限。在这项提议中，我们将通过开发一种新的基于新兴的CRISPR-Cas9基因编辑技术的用于创建DSB的“Break-In”系统来克服这一限制。利用这种方法，我们可以创建对DSB的诱导和修复具有精确的时空控制的DNADSB。破解系统将使我们能够调查当DSB存在时，DNA在3D空间中的组织是如何变化的，以及在损伤修复后，DNA是否返回到其精确的损伤前组织。我们将使用功能分析来确定DNA损伤和DNA组织中的任何相关变化如何导致控制细胞基本特征的基因表达变化。*双链断裂是自发发生的，因此当它们发生时，细胞必须在维持细胞健康的正在进行的细胞过程中管理它们的识别和修复。这项建议的目标是创造一种新的和改进的方法来研究双链断裂，在这种方法中，我们可以了解对双链断裂的反应如何与整体细胞生物学相结合。我们相信，我们的研究将更好地了解DSB如何在功能上影响细胞，以推动细胞身份的变化，以及它们可以在DNA序列本身中造成的变化。**

Environmental agents (e.g. sunlight, naturally occurring radon) and normal metabolism cause DNA damage in all organisms. One of the most dangerous types of DNA damage is the double strand break (DSB) where both sides of the double helix are broken. Repairs of these breaks, while generally accurate, can lead to DNA sequence alterations, chromosome abnormalities or cell death. Our laboratory studies the molecular signaling pathways that sense and repair DSBs to prevent either cell death or permanent changes in the fundamental characteristics of that cell.******Recently, we have studied specifically how these DSB-signaling pathways interact with other cellular processes such as gene expression. To study these pathway interactions, we have engineered several mammalian cell models where we can induce DSBs at specific locations in the DNA. These systems, while valuable models, are limited in the number of locations where DSBs can be produced in cells. In this proposal, we will overcome this limitation by developing a new “Break-IN” system for creating DSBs based on the emerging CRISPR-Cas9 gene editing technology. With this method, we can create DNA DSBs with precise spatial and temporal control of DSB induction and repair. The Break-IN system will allow us to investigate how the organization of DNA in 3D space changes when DSBs are present and if DNA is returned to its precise pre-damage organization following repair of the damage. We will use functional assays to determine how DNA damage and any associated change in the organization of DNA cause alterations in gene expression that control the fundamental characteristics of the cell.******DSBs occur spontaneously, so when they occur cells must manage their recognition and repair in the context of the ongoing cellular processes that maintain cellular health. The goal of this proposal is to create a novel and improved method for studying DSBs where we can learn how the response to DSBs integrates with overall cell biology. We believe that our studies will create a better understanding of how DSBs can impact the cell functionally to drive changes in cellular identity in addition to the changes they can create in the DNA sequence itself. **