Defining the role of PRR12 in cohesin-mediated DNA damage repair and genome stability

About This Grant

Project Summary Each day, cells experience DNA damage from both external factors and normal cellular processes. To overcome this and maintain genomic stability cells rely on multiple cellular programs dedicated to repairing DNA damage. Failure to repair damaged DNA is detrimental to cells and can contribute to many diseases, including developmental disorders and cancer progression. The Cohesin complex plays a key role in the repair of DNA double strand breaks, though the exact mechanism of how Cohesin contributes to this repair is not well understood. Understanding this role has been hindered by the many cellular functions performed by Cohesin, with roles in cell division, transcription, and DNA replication, where disruption to Cohesin leads to broad and indistinguishable phenotypes. Together, this has left an essential gap in our knowledge of how Cohesin is regulated at DNA damage sites and its contribution to disease. Recently, we identified the Cohesin regulator PRR12 that specifically regulates the population of Cohesin at DNA damage sites, however, the mechanisms of this regulation remain to be defined. Understanding the role of PRR12 provides a unique opportunity to specifically target the population of Cohesin at DNA breaks while leaving its other functions intact, an approach that will allow us to specifically address the role of Cohesin in DNA repair for the first time. Additionally, defining the function of PRR12 is critical as patients with mutations in PRR12 phenocopy cohesinopathies, severe developmental and neurological disorders that result from altered Cohesin function. In this proposal, I will define how PRR12 regulates the localization of Cohesin to DNA damage sites and determine the role of the Cohesin complex in efficient DNA repair. To identify how PRR12 interacts with the Cohesin complex and localizes it to DNA lesions I will first perform a structure function analysis of PRR12. Using biochemistry and genetic tools I will determine if this interaction is direct and test its role in Cohesin stabilization. Next, I will determine if Cohesin regulates DNA architecture surrounding DNA breaks using a chromosome conformation capture technique. Last, I will test if PRR12 regulates Cohesin function in two independent DNA repair pathways using a quantitative fluorescent reporter. The work proposed here will be the first to separate the DNA damage role of Cohesin from its various functions throughout the cell cycle and begin to shed light on how misregulation of this process may contribute to genome instability in disease. This training will take place on the CU Medical School campus, where I have access to a wide variety of core resources and opportunities to interact with cell biologist and biochemists in the genome integrity field that will set me up for the successful completion of this proposal. My training plan will expand my experimental toolkit with training in biochemistry and genome organization techniques that will compliment my current expertise in cell biology and microscopy. In addition, I will prioritize gaining the professional development skills, mentorship experience, and communication skills that I will need to transition into an independent research position.