The genetic information in DNA, which is packaged in the form of chromatin, is protected from damage by multiple DNA repair pathways, and understanding how this critical task of DNA repair is performed is the long-term goal of this research. The project will examine real-time changes in chromatin in response to DNA damage in individual living cells with unprecedented spatial and temporal resolution, and thereby determine the significance of chromatin restructuring during DNA repair. In addition to advancing knowledge of DNA damage response and repair mechanisms, the project will develop and apply several innovative technologies that will likely be of great interest to other researchers. The PI and co-PI are committed to disseminating these techniques broadly in the research community. The multidisciplinary nature of this project will provide a unique and valuable training opportunity for graduate students and postdoctoral fellows as well as undergraduate and high school students. They are expected to learn fundamental and cutting-edge techniques relevant to studying DNA repair mechanisms in mammalian cells, including high-resolution imaging.
The chromatin response to DNA damage is highly dynamic and must be regulated to maintain the appropriate architectural organization that promotes high-fidelity DNA repair and preserves genomic integrity. How these chromatin structural dynamics are coordinated with DNA repair is not well understood. The PIs will develop multiplexing novel fluorescence correlation methods including pair correlation spectroscopy (pCF) and nanoimaging tracking techniques to analyze how DNA damage affects chromatin dynamics and how that in turn dictates DNA repair pathway choice. These phenomena will be investigated in response to relatively simple DNA breaks or complex DNA damage lesions induced in different cellular contexts. Aim 1 includes monitoring changes in chromatin access at the damage site, interrogating the relationship to DNA repair pathway choice, and determining responsible factors. Aim 2 involves high-resolution multiplexed analyses of chromatin dynamics at defined DNA double-strand break sites, and investigating the interplay between DNA repair and transcription. An overall goal is also to develop new methods that yield 4D and nanoscale views of chromatin dynamics in single mammalian cells when DNA is damaged, for a quantitative understanding of how the cellular machinery assesses the damage and decides how to repair its genome.
