Cell cycle regulation of DNA double-strand break end resection by Cdk1-dependent Dna2 phosphorylation

Abstract

DNA recombination pathways are regulated by the cell cycle to coordinate with replication. Cyclin-dependent kinase (Cdk1) promotes efficient 5' strand resection at DNA double-strand breaks (DSBs), the initial step of homologous recombination and damage checkpoint activation. The Mre11-Rad50-Xrs2 complex with Sae2 initiates resection, whereas two nucleases, Exo1 and Dna2, and the DNA helicase-topoisomerase complex Sgs1-Top3-Rmi1 generate longer ssDNA at DSBs. Using Saccharomyces cerevisiae, we provide evidence for Cdk1-dependent phosphorylation of the resection nuclease Dna2 at Thr4, Ser17 and Ser237 that stimulates its recruitment to DSBs, resection and subsequent Mec1-dependent phosphorylation. Poorly recruited dna2T4A S17A S237A and dna2ΔN248 mutant proteins promote resection only in the presence of Exo1, suggesting cross-talk between Dna2- and Exo1-dependent resection pathways.

Figure 1

Cdk1 regulates long range DSB resection by Dna2. (a) Analysis of initial and extensive resection in mutants with either active or inactive Cdk1 kinase. The Southern blots corresponding to these experiments are presented in Supplemental Figure 1. Error bars correspond to s.d. (b) Southern blot analysis of initial resection in mutants. Smearing and additional bands below the HO cut band typical for mutants that lack extensive resection are indicated by asterisks. (c) Analysis of resection in cells where Cdk1 activity is blocked at 4 hours after break induction when all cells have initiated resection. (d) Recruitment of Dna2 to DSBs was monitored in indicated mutants by ChIP. Error bars correspond to s.d.

Figure 2

Dna2 is phosphorylated by Cdk1 and Mec1. (a) Western blot analysis of Dna2-9×Myc phosphorylation in cdk1-as1 with or without Cdk1 inhibitor and in checkpoint deficient cells in response to a single DSB. (b) In vitro phosphorylation of wild-type Dna2 or dna2 mutant proteins lacking single or multiple Cdk1 phosphorylation consensus sites. (c) Western blot analysis of Dna2 phosphorylation in wild type cells and indicated dna2 mutants cells.

Figure 3

Dna2 phosphorylation by Cdk1 stimulates resection. (a) Analysis of 5' strand resection in dna2Δ cells complemented with plasmids carrying either wild-type or a mutant DNA2 allele. Error bars correspond to s.d. The Southern blots corresponding to these experiments are presented in Supplemental Figure 3. (b) Analysis of resection in exo1Δ dna2Δ cells complemented with plasmids carrying either wild-type or a mutant DNA2 allele. (c) Analysis of sensitivity to MMS and camptothecin in the indicated mutants.

Figure 4

Dna2 phosphorylation by Cdk1 is needed for its recruitment to DSBs. (a) Analysis of DSB recruitment of GFP-tagged wild-type Dna2 and indicated mutant dna2 proteins. (b) Analysis of recruitment of FLAG-tagged wild-type Dna2 and indicated mutant dna2 proteins to DSB ends by ChIP using primers specific for sequences located 1 kb upstream of the DSB. Error bars correspond to s.d. (c) Analysis of recruitment of GFP-tagged phosphomimic dna2^{S17D S237D} protein to DSB ends in Cdk1 and Ku deficient cells.