Damage to our DNA poses one of the most direct threats to both the ability of our cells to function normally, as well as our ability to faithfully transmit genetic information to our progeny. Our cells experience a significant amount of spontaneous DNA lesions. Life, as we know it, is possible thanks to specific DNA repair mechanisms that eukaryotic cells have evolved. Such mechanisms involved the highly coordinated action of sensors, amplifiers and effectors that work in concert with signaling pathways to allow efficient DNA repair, in what is known as the DNA Damage Response (DDR). Our DNA is densely packed in chromatin, a barrier that needs to be overcome for proper recognition and repair of DNA lesions. Indeed, chromatin dynamics has emerged as an important module in the DDR, however the specific chromatin factors that are directly involved in the repair process remain poorly known. In this context, advances in the field have been hampered by limited availability of high-throughput technologies. In this exploratory grant, we will attempt to tackle these limitations by developing both a library of chromatin factors and a microscope based-high throughput assay to follow kinetics and recruitment of chromatin factors to DNA breaks. Specifically, in Aim 1 we will develop a library (ChromORFeome) of chromatin factors and use a high-throughput laser breaks assay to identify novel chromatin factors recruited to sites of DNA breaks.
Aim 2 will provide proof of principle validation of these novel identified chromatin factors. Results from this exploratory grant will establish advanced new technologies to study the process of DNA repair and identify novel chromatin factors that play key roles in this process.
Our cells are constantly exposed to spontaneous insults that generate DNA lesions, which if unrepaired, could lead to gene mutations that drives numerous diseases, including cancer, neurodegeneration and immunologic syndromes. Higher organisms have evolved sophisticated mechanisms of DNA repair that support life as we know it. In this regard, our DNA is packed inside our cells in a compact structure termed chromatin, and such structure needs to undergo dynamic changes in order for repair factors to efficiently fix DNA lesions. In this proposal, we aim to develop a novel high-throughput methodology to identify key chromatin factors involved in DNA repair.
