Spatiotemporal regulation of DNA metabolism pathways

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Spatiotemporal Regulation of DNA Metabolism Pathways PROJECT SUMMARY/ABSTRACT Myriad DNA lesions occur continuously, and they necessitate distinct DNA damage repair mechanisms for removal. DNA double stranded breaks (DSBs) are potentially deleterious lesions that can trigger extensive loss of genetic information, chromosome fusions, and other gross chromosome rearrangements. DSBs are repaired mostly by homology-driven (Homologous Recombination: HR) or homology-independent (Non-Homologous End Joining, NHEJ) mechanisms. The choice of the repair pathway is dictated by the cell cycle phase, with HR being the more accurate (conservative) mechanism. Pathologies, such as meiotic defects and infertility, developmental syndromes, and cancer could stem from defects in HR. In addition to their involvement in DSB repair, many HR proteins fulfill key roles in the resolution of stalled replication forks or difficult-to-replicate DNA structures such as centromeres, telomeres, or DNA-RNA hybrids (R-loops). Importantly, HR proteins must be selectively activated to only perform repair functions at DNA lesions and differentially regulated to fulfill their crucial functions at other DNA structures such as stalled or collapsed replication forks. Timely activation/deactivation of HR proteins is thus a pre-requisite for maintaining genomic stability and avoidance of pathologies. There is a major gap of knowledge in understanding how the activity of HR proteins is dampened at DNA structures that resemble DNA lesions but activated at pathological structures, and how they fulfill unique roles at DNA breaks and replication forks. We postulate that phosphorylation and dephosphorylation of tyrosine residues, an under-studied subject as compared to serine/threonine modifications, contribute to the dynamic regulation of HR proteins. In our effort to fill this crucial knowledge gap, we have provided compelling evidence that EYA4, a dual activity protein phosphatase, acts on key HR and NHEJ factors to exert seminal impact on DNA repair efficiency and pathway choice. Over the past several years, we have devised biochemical procedures for the expression and purification of EYA4, and have identified RAD51 and 53BP1, central components of HR and NHEJ, respectively, as substrates of this poorly characterized protein phosphatase. We have made considerable progress in delineating the contributions of key phospho-residues in RAD51 to its role in HR, and in 53BP1 to NHEJ. We will now conduct mechanistic studies to understand the cellular regulation of EYA4, and whether its role in DNA repair pathway choice impacts the preservation of stressed and damaged replication forks and affects the maintenance of telomeres.