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

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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. **