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

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Abstract
DNA can be repaired by two fundamentally different mechanisms, depending on whether a homologous template is available or not. Given that DNA is duplicated in S phase, homologous recombination is restricted to S and G2 phases of the cell cycle. The activity of cyclin-dependent kinases (CDKs) is also cell-cycle regulated, and the yeast CDK Cdc28 controls DNA resection, an early step of homologous recombination. In this work, Huertas et al. show that the target of Cdc28 in regulating DNA resection is Sae2, a protein with endonuclease activity that was first identified as being required for meiotic recombination. These results support models in which the commitment to DSB resection is highly regulated to ensure that the cell engages the most appropriate DNA repair pathway at the right time, thereby optimizing genome stability. DNA can be repaired by two different mechanisms, depending on whether a homologous template is available. Thus, homologous recombination is restricted to S and G2 phases of the cell cycle. The activity of cyclin-dependent kinases (CDKs) is also cell cycle regulated, and the yeast CDK Cdc28 controls DNA resection, an early step of homologous recombination. This work shows that the target of Cdc28 in regulating DNA resection is Sae2. 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 template2,3,4,5. 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 cell-cycle 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.