CDK targets Sae2 to control DNA-end resection and homologous recombination

Abstract

DNA double-strand breaks (DSBs) are repaired by two principal mechanisms: non-homologous end-joining (NHEJ) and homologous recombination (HR) 1 . HR is the most accurate DSB repair mechanism but is generally restricted to the S and G2 phases of the cell cycle, when DNA has been replicated and a sister chromatid is available as a repair template [2][3][4][5] . By contrast, NHEJ operates throughout the cell cycle but assumes most importance in G1 (refs 4, 6). The choice between repair pathways is governed by cyclin-dependent protein kinases (CDKs) 2,3,5,7 , with a major site of control being at the level of DSB resection, an event that is necessary for HR but not NHEJ, and which takes place most effectively in S and G2 (refs 2, 5). Here we establish that cellcycle control of DSB resection in Saccharomyces cerevisiae results from the phosphorylation by CDK of an evolutionarily conserved motif in the Sae2 protein. We show that mutating Ser 267 of Sae2 to a non-phosphorylatable residue causes phenotypes comparable to those of a sae2Δ null mutant, including hypersensitivity to camptothecin, defective sporulation, reduced hairpin-induced recombination, severely impaired DNA-end processing and faulty assembly and disassembly of HR factors. Furthermore, a Sae2 mutation that mimics constitutive Ser 267 phosphorylation complements these phenotypes and overcomes the necessity of CDK activity for DSB resection. The Sae2 mutations also cause cell-cycle-stage specific hypersensitivity to DNA damage and affect the balance between HR and NHEJ. These findings therefore provide a mechanistic basis for cell-cycle control of DSB repair and highlight the importance of regulating DSB resection.To initiate HR, one strand of the broken DNA duplex is resected in the 5′→3′ direction, generating single-stranded DNA (ssDNA) that can anneal with a homologous DNA duplex 8 . In S. cerevisiae, effective resection and HR require sustained Cdc28/Clb (Cdk1/cyclin B) kinase activity 2,3,5 , although the CDK targets mediating this control are still unknown. One potential target is Sae2, a protein first identified as being required for meiotic recombination. Sae2 controls the initiation of DNA-end resection in meiotic and mitotic cells [9][10][11][12] and was recently shown to be a DNA endonuclease 13 Fig. 2b and Supplementary Fig. 2a). In accord with this idea, the amount of slower-migrating Sae2 was diminished when Cdc28 was inactivated in G2-synchronized cultures by galactose-driven expression of the Cdc28/Clb repressor, Sic1 (ref. 15) (Fig. 1a).Sae2 contains three potential CDK phosphorylation sites ( Fig. 1b and Supplementary Fig. 1); two of these-Ser 267 and Ser 134-received the highest scores for predicted phosphorylation sites in the protein (Supplementary Table 1). Ser 267 maps to the Sae2 region most highly conserved with its non-yeast orthologues (Fig. 1b), which include human CtIP, Caenorhabditis elegans Com1 and Arabidopsis thaliana Com1 (refs 16-18). To address the possible function(s) of Ser 267 and other potential target site...

Cdk-mediated phosphorylation of Chk1 is required for efficient activation and full checkpoint proficiency in response to DNA damage

“…Cdk phosphorylation regulates Chk1 activation N Xu et al that Cdk activity has a major role in controlling damage processing (Huertas et al, 2008;Wohlbold and Fisher, 2009). As a result of its central role in initiating both checkpoint signaling (via ATR activation) and DNA repair by homologous recombination, we postulated that DNA damage-induced strand resection might exhibit a similar pattern of cell cycle regulation to Chk1 activation.…”

“…The first step is Cdk2-mediated phosphorylation of CtIP, which stimulates DSB resection and thus determines the strength of ATR activation during the cell cycle (Huertas et al, 2008;Huertas and Jackson, 2009). Other Cdk targets might also be involved in DSB resection but the contribution of these is not as clear as for CtIP (Wohlbold and Fisher, 2009;Yata and Esashi, 2009).…”

“…A recently identified Cdk target involved in DNA strand resection at DSBs is Sae2, a homolog of CtIP in vertebrates and Ctp1 in fission yeast. Cdk-dependent phosphorylation of Sae2 at Ser267 is needed for proper DNA end-resection and HRR (Huertas et al, 2008). In mammalian cells, Cdks have a role in DSB resection via CtIP.…”

The plant‐specific CDKB 1‐ CYCB 1 complex mediates homologous recombination repair in Arabidopsis

“…An early and decisive step in HR is the resection of the 3′ ends at the site of the DSB to allow subsequent strand invasion of the damaged into the undamaged chromatid. In animals and yeast, it has been found that CDKs phosphorylate proteins in the MRN complex that process 3′ ends, for example, Nbs1, and proteins that work in concert with this complex, such as the nuclease Sae2/CtIP/Com1 (Huertas et al , 2008; Huertas & Jackson, 2009; Wohlbold et al , 2012; Simoneau et al , 2014). Conversely, mutants of Nbs1 in which the phosphorylation site of Cdk2 was eliminated were hypersensitive to radiation in a similar manner as cells in which Cdk2 was chemically inhibited (Wohlbold et al , 2012).…”

Regulation of Recombination and Genomic Maintenance

“…The complexity of the DSB resection machineries has only recently been established by genetic and biochemical approaches (see Symington 2014). A key conserved target for cell-cycle-dependent kinases in the end-resection machinery was identified as yeast Sae2 and its mammalian homolog CtIP (Huertas et al 2008;Huertas and Jackson 2009). Sae2/CtIP cooperate with the Mre11/Rad50/Xrs2 (mammalian MRE11/ RAD50/NBS1) complex to provide the initial resection of DSBs.…”