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

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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 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.

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Figure 1: Ser 267 mutation impairs Sae2 function.
Figure 2: Sae2 is phosphorylated by Cdc28 on Ser 267.
Figure 3: DNA-end resection is controlled by Sae2.
Figure 4: Sae2 mutations affect Mre11 and Rad52 dynamics, and DSB repair.

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Acknowledgements

We thank M. P. Longhese, R. Rothstein, K. Lobachev, M. Lichten and M. Foiani for providing strains, and R. Driscoll, S. Gravel, K. Dry and K. Miller for helpful discussions and comments on the manuscript. P.H. is the recipient of a Long-Term EMBO Fellowship. A.A.S. is supported by a Swiss National Foundation Grant. The S.P.J. laboratory is supported by grants from Cancer Research UK and the European Community (Integrated Project DNA repair, grant LSHG-CT-2005-512113). The A.A. laboratory is supported by grants from the Spanish Ministry of Science and Education (BFU2006-05260 and CDS2007-0015) and Junta de Andalucia (CVI624).

Author Contributions A.A.S. identified the homology between Sae2 and CtIP, cloned SAE2 into pGEX-4T1 and made the original sae2-S267A and sae2-S267E mutations. All the experiments shown were performed by P.H. and were conceived by P.H. and S.P.J., except those on SCR analyses that were performed by F.C.-L. and conceived by F.C.-L. and A.A. P.H. and S.P.J. wrote the paper. All authors discussed and commented on the manuscript.

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Correspondence to Stephen P. Jackson.

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