Coordination of Rad1-Rad10 with Msh2-Msh3 and Saw1 in the initiation of 3' non-homologous tail removal
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3' non-homologous tail removal (3'NHTR) is a required step in certain forms of double strand break repair (DSBR). The process of 3'NHTR is unique in that all of the proteins required for 3'NHTR also function in two or more DNA repair pathways. In Saccharomyces cerevisiae, the protein components required for 3'NHTR have been identified but their exact function how proteins are coordinated together in 3'NHTR is not known. Rad1-Rad10 is a structure specific endonuclease that cleaves the 3'non-homologous tails (3'NHT). Msh2-Msh3 binds and stabilizes the 3'NHTs while also interacting with Rad1-Rad10. Saw1 was recently identified in a genetic screen and was determined to interact with Rad1-Rad10 and Msh2-Msh3. Slx4 mediates cross-talk with the DNA damage response while also interacting with Rad1-Rad10. In this work we have studied the protein players of 3'NHTR to further the understanding of their function and coordination in 3'NHTR. We first examined the localization requirements of Rad1-Rad10 in 3'NHTR. We determined that Saw1 and Msh2-Msh3 are required for the localization step and that Slx4 functions in a later step in the pathway. Through this work, we demonstrated that Saw1 is essential for the localization of Rad1-Rad10 to the recombination intermediate. Saw1 forms a stable complex with Rad1-Rad10 and recruits Rad1-Rad10 through protein-protein and protein-DNA interactions to the 3'NHT and enhances the endonuclease activity of Rad1-Rad10. We next examined the coordination between Rad1-Rad10, Msh2-Msh3 and Saw1 utilizing separation-of-function mutations of Rad1. We found that the process of 3'NHTR requires balanced interactions between Msh2-Msh3, Rad1-Rad10, and Saw1. We determined that the rad1 mutants had altered interactions with either Msh2-Msh3 or Saw1 or both in vitro. We examined the effects of altering the protein levels of Msh2-Msh3 and Saw1 in vivo with one of the rad1 alleles. We found that increased amounts of Saw1 increased survival while increased amounts of Msh2-Msh3 decreased survival. The in vitro and in vivo data support the hypothesis that the coordination of Rad1-Rad10 with Msh2-Msh3 and Saw1 is tightly regulated in order to allow for proper function of 3'NHTR.