DNA end resection, homologous recombination and DNA damage checkpoint activation require CDK1

Abstract

A single double-strand break (DSB) induced by HO endonuclease triggers both repair by homologous recombination and activation of the Mec1-dependent DNA damage checkpoint in budding yeast. Here we report that DNA damage checkpoint activation by a DSB requires the cyclin-dependent kinase CDK1 (Cdc28) in budding yeast. CDK1 is also required for DSB-induced homologous recombination at any cell cycle stage. Inhibition of homologous recombination by using an analogue-sensitive CDK1 protein results in a compensatory increase in non-homologous end joining. CDK1 is required for efficient 5' to 3' resection of DSB ends and for the recruitment of both the single-stranded DNA-binding complex, RPA, and the Rad51 recombination protein. In contrast, Mre11 protein, part of the MRX complex, accumulates at unresected DSB ends. CDK1 is not required when the DNA damage checkpoint is initiated by lesions that are processed by nucleotide excision repair. Maintenance of the DSB-induced checkpoint requires continuing CDK1 activity that ensures continuing end resection. CDK1 is also important for a later step in homologous recombination, after strand invasion and before the initiation of new DNA synthesis.

Figure 1

CDK1 activity is required for DSB-induced phosphorylation of checkpoint proteins in G2 cells. The phosphorylation of checkpoint proteins in the presence of an HO-induced unrepaired DSB in G2/M cells arrested with nocodazole (N) is shown, comparing cells with active CDK1 or GAL::SIC1-inactivated CDK1.

Figure 2

CDK1 is required for homologous recombination. a, MAT switching is initiated by creating an HO-induced DSB at the MAT locus that is repaired by gene conversion from HML or HMR. MAT switching is shown in asynchronous cells or cells arrested in G1 (α-factor arrest) or G2/M (nocodazole arrest). b–d, MAT switching in cdc28-as1 (b), wild-type (c) and cdc7-as3 (d) strains with and without 1-NMPP1 inhibitor. e, Allelic recombination in a cdc28-as1 homozygous diploid strain. The position of two DNA probes is shown: MAT-distal (MD, verifies DSB induction) and MAT-proximal (MP, detects product). f, Efficiency of NHEJ in cdc28-as1 cells with either active or inactive CDK1 at different stages of the cell cycle. Error bars indicate s.d.

Figure 3

CDK1 is needed for DSB resection. a, The 5′ to 3′ resection of DSB ends was analysed in the indicated mutants and at different points in the cell cycle by loss of the HO-cut EcoRI restriction fragment in a strain lacking HML or HMR. b, ChIP analysis of binding of RPA (left), Rad51 (centre) and Mre11 (right) to DSB ends in a donorless strain^,, in asynchronous or G1 cells and in G2/M-arrested cells with or without Sic1 inhibition of CDK1. Where error bars are shown, results are means ± s.d. c, Lack of Rad53 phosphorylation in mre11Δ G2/M-arrested cells, compared with other conditions.

Figure 4

CDK1 is required to maintain Rad53 activation in response to a DSB but not after damage by 4NQO. a, In G1-arrested cells, Rad53 is not phosphorylated when an HO-mediated DSB is induced by galactose, but is triggered when 4NQO provokes nucleotide excision repair. In nocodazole-arrested G2/M cells, treatment with 4NQO causes Rad53 phosphorylation even when CDK1 is inhibited by Sic1. b, At 6 h after DSB induction, when the checkpoint is established and Rad53 is phosphorylated, 1-NMPP1 was added to either CDC28 or cdc28-as1 strains. c, Rad53 phosphorylation is lost when CDK1 is inactivated, and resection of a StyI fragment, 20 kb from the DSB, is inhibited. Open triangles, cdc28-as1 G2 + 1-NMPP1, filled triangles, cdc28-as1 G2, open circles, wild type G2 + 1-NMPP1.

Figure 5

Role of CDK1 in a late stage of MAT switching. a, Cdc28-as1 was inhibited in cells at different times before and after HO induction, and the amount of product formed at 6 h was determined. b, Strand invasion was tested by ChIP analysis for RPA and Rad51 in a cdc28-as1 strain. The diagram shows homologous sequences (dashed outlined boxes) and primers used to detect immunoprecipitation (IP) of MAT (p1, p2) or HML (p1, p3) with anti-Rad51 or Rfa1 antibodies. Symbols indicate the time at which inhibitor was added with respect to break induction: triangles, no inhibitor; squares, 0.5 h; circles, 1.5 h. c, Polymerase chain reaction detects the first new DNA synthesis primed by the 3 ′ invading end after strand invasion. Circles, no inhibitor; triangles, 1-NMPP1 added 1.5 h after HO induction. Where error bars are shown, results are means ± s.d.