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). 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. 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), 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 sae2Delta 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.

Figure 1. Ser 267 mutation impairs Sae2 function

a, Left: TAP-tagged Sae2 was purified from cells expressing galactose-inducible SIC1 (Gal) or not expressing SIC1 (Glc). U, control untagged strain. Right: as above, but the strain lacked galactose-inducible SIC1. b, Sae2 diagram and homology to orthologues (see Methods for full alignment). S. cerevisiae, Saccharomyces cerevisiae, A. gossypii, Ashbya gossypii; Y. lipolytica, Yarrowia lipolytica; C. neoformans, Cryptococcus neoformans; N. crassa, Neurospora crassa; C. globosum, Chaetomium globosum; P. nodorum, Phaeosphaeria nodorum; C. elegans, Caenorhabditis elegans; A. thaliana, Arabidopsis thaliana; Xenopus, Xenopus laevis; chicken, Gallus gallus; human, Homo sapiens. c, Fivefold serial dilutions of sae2Δ cultures containing the indicated SAE2 genes plated on medium lacking or containing camptothecin (5 μg ml⁻¹). d, Extracts of cells harbouring TAP-tagged Sae2 variants were western immunoblotted as indicated. e, Survival of U2OS cells expressing GFP–CtIP fusions to 1 h treatments with the indicated doses of camptothecin. Error bars indicate s.d. (n = 2).

Figure 2. Sae2 is phosphorylated by Cdc28 on Ser 267

a, TAP-tagged Sae2 derivatives were immunoprecipitated and detected as indicated. b, TAP-tagged Sae2 was purified from G1 or G2 arrested cultures. U, G2 arrested untagged control cells. Immunoprecipitated samples and inputs (5%) were immunoblotted as indicated. c, Glutathione S-transferase (GST)-fused Sae2 and Sae2-S267A were purified, incubated with recombinant Cdk2/Cyclin A and ATP, resolved by 10% SDS–PAGE and immunoblotted as indicated. d, Recombination frequencies of strains in a hairpin-containing recombination system. e, Spores after 24 h in sporulation medium. f, Spore viability 24 h after the addition of sporulation medium (SPM). Error bars in d-f represent s.d. (n = 2).

Figure 3. DNA-end resection is controlled by Sae2

a, Resection-mediated ssDNA formation at an HO DSB in wild-type SAE2 (filled squares), sae2-S267A (open triangle), sae2-S267E (open diamonds) or empty vector (solid circles) at indicated times after HO induction at the MAT locus (left), 5 kb downstream of MAT (centre) or LEU2 locus (right). b, Wild-type SAE2 (squares) or sae2-S267E (diamonds) strains containing Cdc28-as1 were grown as in a but in the presence of dimethylsulphoxide (filled symbols) or 1NM-PP1 (open symbols). Results are shown as means ± s.d. (n = 5).

Figure 4. Sae2 mutations affect Mre11 and Rad52 dynamics, and DSB repair

a, b, Percentages of S/G2 cells containing Mre11 (a) or Rad52 (b) foci. c, Percentage of G1 cells containing a Rad52 focus. d, Survival of sae2Δ mutants containing wild-type SAE2, sae2-S267A, sae2-S267E or empty vector grown asynchronously (Async) or arrested in G1 or G2, after irradiation with 30 Gy. Error bars represent s.d. (n = 2). e, Sister-chromatid recombination measured as described previously. Standard deviations of two independent experiments are shown (see Supplementary Fig. 4 for details and representative blot). f, Plasmid cleaved by Xho I, Sac I or Sma I was transformed into strains and NHEJ efficiency was measured. Means and s.d. of three independent experiments are shown. g, Classes of plasmid rejoining products from 50 independent clones of each strain transformed with the Xho I-cut plasmid.