Maintaining genome stability is essential for cell viability and growth. Post-translational modifications such as phosphorylation have been implicated in regulating the cellular processes that maintain genome integrity. Under genotoxic stress, cells initiate a DNA damage response that includes the activation of a phosphorylation-mediated pathway known as the DNA damage checkpoint. Several processes are regulated by the DNA damage checkpoint including cell cycle arrest, DNA repair, transcription, and cell apoptosis. In Saccharomyces cerevisiae, Mec1 and Tel1 are key protein kinases that initiate the activation of the DNA damage checkpoint. Several studies have identified substrates of Mec1/Tel1 including Sae2, a DNA double-strand break repair protein involved in DNA end resection and processing of hairpin structures. In this study we identify Mec1/Tel1 consensus sites on Sae2, T90 and T279, which when mutated to non-phosphorylatable alanine causes genome instability and hyper-activation of the downstream checkpoint kinase Rad53, similar to a sae2∆ mutant. We find that phosphorylation of T90 and T279 on Sae2 mediates protein-protein interaction with several Forkhead associated-domain containing proteins including checkpoint kinases Rad53 and Dun1. Taken together, this suggests that the interaction of phosphorylated Sae2 with Rad53 and Dun1 is important for its role in DNA repair and genome maintenance.
Pathways that regulate other post-translational modifications have also been implicated in genome stability, including the small ubiquitin-like modifier (SUMO). Recently, SUMO E3 ligases have been shown to be suppressors of gross chromosomal rearrangements, but not much is known of the enzymes that deconjugate SUMO. In this study, we discover a role for SUMO isopeptidases, Ulp1 and Ulp2, in preventing gross chromosomal rearrangements. We identify targets of Ulp1 and Ulp2 and find that Ulp2 distinctly regulates sumoylation at three chromosomal regions: rDNA, centromeres, and origins of replication. In contrast, Ulp1 globally targets most sumoylated proteins and mutating ULP1 leads to an unexpected decrease in sumoylation of Ulp2 substrates. Moreover, we find that Ulp2 is able to target its substrates at the rDNA and centromeres through interaction with a kinetochore-associated complex Csm1-Lrs4. Structural and biochemical analysis demonstrates that the C-terminus of Ulp2 binds to the globular domain of the Csm1 homodimer. Mutations to the residues on Ulp2 that interact with Csm1 elevate sumoylation of the nucleolar protein Tof2 and reduce its protein abundance, resulting in a loss of transcriptional silencing at the rDNA. Lastly, we show that the loss of Tof2 protein levels in ulp2 mutants are triggered by the ubiquitin E3 ligase complex, Slx5-Slx8.