An appropriate cellular response to DNA damage, which includes damage recognition, cell cycle checkpoint activation, and repair, is crucial for the maintenance of genome integrity. Since perturbation of this response can jeopardize cell function and survival, it is important to understand the underlying molecular mechanism. Cohesin is an essential multiprotein complex that associates with DNA during interphase and is involved in the establishment and maintenance of sister chromatid cohesion. Genetic evidence suggests that cohesin also plays a role in DNA double strand break (DSB) response/repair. However, it was unclear whether this was due to general perturbation of cohesion, or whether cohesin has a specialized role at the damage site. Recently, my laboratory has developed a method to introduce damage at a discrete site in the cell nucleus using laser microirradiation, allowing subsequent immunofluorescent detection of factors recruited to the damage site. This system provides a unique tool to study the kinetics of, and the requirement(s) for, factor recruitment/modification at the sites of DNA damage in vivo. Using this system, we demonstrated that human cohesin is recruited to the damage in both an S/G2-specific and the DSB repair factor Mrel l/Rad50- dependent manner. Based on these results, we hypothesize that cohesin has a specialized role in cell cycle- specific DSB response/repair, most likely in homologous recombination repair (HR), by establishing the local sister chromatid cohesion at the damage sites. The objective of this project is Loexamine the mechanism of cohesin recruitment to DSB damage to address its role at the damage sites.
Specific aims are 1) analysis of cohesin recruitment to DNA damage using the laser system complemented by chromatin crosslinking and immunoprecipitation (CHIP) of restriction enzyme-induced DNA breaks, 2) biochemical dissection of the domain(s) within the cohesin complex that is required for DNA damage recognition/targeting, and 3) identification and characterization of proteins that dictate DNA damage recognition by cohesin, including the factors important for the establishment of sister chromatid cohesion for mitosis, using RNAi and/or mutant cells and protein interaction assays. Understanding the role of cohesin in DNA repair will provide an important insight into the mechanism of the DNA damage response in mammalian cells and may provide a novel marker or a potential therapeutic target for cancer diagnosis and treatment.
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