Regulatory Mechanisms Of Rad55 Rad57 That Influence Presynaptic Filament Formation During Homologous Recombination

The proper assembly of Rad51 recombinase into a polymeric filament on single-stranded DNA marks a critical juncture in the process of homologous recombination. A functional Rad51 nucleoprotein filament is capable of performing the trademark reactions of homologous recombination: homology search and DNA strand exchange. A number of accessory factors, termed mediators, are essential to promote the assembly of Rad51 filaments. In Saccharomyces cerevisiae these genes comprise members of the RAD52 epistasis group including its namesake member RAD52, RAD55 and RAD57. The process of Rad51 binding to single-stranded DNA occurs in competition with other repair pathways (e.g. non-homologous end-joining, single-strand annealing, translesion synthesis) and proteins (e.g. RPA, Srs2) that either inhibit or actively disassemble Rad51 filaments. These processes are highly dynamic and ultimately contribute to determining which repair pathway is utilized. Events that govern repair pathway choice have been intensively studied over the past decade; however, the mechanisms specifically regulating homologous recombination remain relatively poorly understood. Previous work established that in response to DNA damage Rad55 is phosphorylated by kinases of the DNA damage response pathway, and this serves to promote the function of Rad55 as a mediator during homologous recombination. Here, these findings are extended with evidence that Rad55 phosphorylation promotes its activity in part by stabilizing the protein against degradation. Additionally, an epistatic miniarray profile (EMAP) analysis was used as an unbiased, high-throughput method to screen for novel mechanisms of Rad51 filament regulation. This screen led to the discovery of a novel connection between proteins that function in nonsense-mediated decay and Rad51 filament formation. Further investigation revealed that nonsense-mediated decay affects Rad51 filament formation by controlling RAD55 mRNA levels. The various aspects of Rad55 regulation uncovered over the course of these studies are synthesized into a working model, and the emerging picture of a highly dynamic and complex regulatory network and its implications for the process of DNA repair pathway choice are discussed.