A screen for synthetic phenotypes reveals new Sae2 functions and interactions in the repair of DNA double-strand breaks

Genome instability is one of the most pervasive characteristics of cancer cells and can be due to DNA repair defects and failure to arrest the cell cycle. Among the many types of DNA damage, the DNA double strand break (DSB) is one of the most severe, because it can cause mutations and chromosomal rearrangements. Generation of DSBs triggers a highly conserved mechanism, known as DNA damage checkpoint, which arrests the cell cycle until DSBs are repaired. DSBs can be repaired by homologous recombination (HR), which requires the DSB ends to be nucleolytically processed (resected) to generate single-strand DNA. In Saccharomyces cerevisiae, initiation of DSB resection requires the conserved MRX/MRN complex (Mre11-Rad50-Xrs2 in yeast; Mre11-Rad50-Nbs1 in mammals) that, together with Sae2 (CtIP in mammals), catalyzes an endonucleolytic cleavage of the 5’ strands. More extensive resection depends on two pathways: one catalyzed by the exonuclease Exo1, and a second requiring the nuclease Dna2 with the helicase Sgs1. The absence of Sae2 not only impairs DSB resection, but also causes prolonged MRX binding at the DSBs that leads to persistent Tel1 (ATM in humans)- and Rad53-dependent DNA damage checkpoint activation and cell cycle arrest. Whether this enhanced checkpoint signaling contributes to the DNA damage sensitivity and/or the resection defect of sae2∆ cells is not known. sae2∆ cells are sensitive to the alkylating agent methyl methanesulfonate (MMS) and camptothecin (CPT), which traps covalent topoisomerase I (Top1)-DNA cleavable complexes and induces DNA replication-dependent cell death. Since this sensitivity has been shown to be due to resection defect, we searched for extragenic suppressors of the sae2∆ sensitivity to CPT and MMS. By performing a genetic screen, we identify three mutant alleles (SGS1-ss, rad53-ss and tel1-ss) that suppress both the DNA damage hypersensitivity and the resection defect of sae2∆ cells. We show that Sgs1-ss mediated suppression depends on the Dna2 nuclease but not on Exo1. Furthermore, not only Sgs1-ss suppresses the resection defect of sae2∆ cells but it also increases resection efficiency compared to wild type cells. The checkpoint protein Rad9 limits the action of Sgs1/Dna2 in DSB resection by inhibiting Sgs1 binding/persistence at the DSB ends. When inhibition by Rad9 is abolished by the Sgs1‐ss mutant variant or by deletion of RAD9, the requirement for Sae2 and functional MRX in DSB resection is reduced. rad53-ss and tel1-ss mutant alleles, but also the kinase defective alleles (rad53-kd and tel1-kd), suppress both the DNA damage hypersensitivity and the resection defect of sae2∆ cells through an Sgs1-Dna2-dependent mechanism. These suppression events do not involve escaping the checkpoint-mediated cell cycle arrest. Rather, defective Rad53 or Tel1 signaling bypasses Sae2 function at DSBs by decreasing the amount of Rad9 bound at DSBs. As a consequence, reduced Rad9 association to DNA ends relieves inhibition of Sgs1-Dna2 activity, which can then compensate for the lack of Sae2 in DSB resection and DNA damage resistance. We propose that persistent Tel1 and Rad53 checkpoint signaling in cells lacking Sae2 cause DNA damage hypersensitivity and defective DSB resection by increasing the amount of Rad9 bound at the DSBs, which in turn inhibits the Sgs1-Dna2 resection machinery.