Fig. 2: DDK promotes DNA end resection and phosphorylates resection nucleases.

a A DSB generated by the HO endonuclease in the LEU2 open reading frame can be repaired via single-strand annealing (SSA) upon extensive resection that reveals a region of homology 25 kb distant from the DSB, followed by annealing of the homologous sequences and ligation. RAD51 is deleted to suppress HR. b Five-fold serial dilutions of reported strains were spotted on YPD (as control) or YPGal (for chronic pGAL-HO-induction) plates. Data are representative of n = 3 biological replicates. c Strand-specific loss of DNA and enrichment of RPA-bound ssDNA is used to measure resection at an unrepairable DSB, generated by the HO endonuclease at the MAT locus. d, e DDK is required for efficient DNA end resection. d Total DNA shows preferential loss of 5′ DNA strands 2 and 4 h after DSB induction. Loss of DNA is less pronounced after IAA-induced degradation of Dbf4-3AID. Strand-specific read coverage is normalized to read coverage before DSB induction. e Strand-specific accumulation of 3′-ssDNA enriched by RPA-ChIP is diminished after IAA-induced degradation of Dbf4-3AID compared to mock-treated cells. RPA-ChIP signals at the DSB are normalized to DSB-independent RPA signals occurring throughout the genome. Data are representative of n = 4 biological replicates. See also Supplementary Fig. 2e, f. f DDK inhibition leads to defective resection in human cells. M-phase-arrested U2OS cells were treated with the DDK inhibitor XL413 (or mock-treated) before DNA damage induction with zeocin (or mock-treatment). RPA foci (RPA70 subunit) were counted as proxy for resection. Left, representative images of cells (DAPI, RPA70). Scale bars: 10 μm. Right, quantification of RPA foci number per cell. Blue bars represent the mean. Reported p-values were calculated using a two-tailed Mann-Whitney test. Mock/mock n = 171 cells, mock/zeocin n = 230 cells, XL413/mock n = 169 cells, XL413/zeocin n = 265 cells; pooled from n = 2 biological replicates. See also Supplementary Fig. 2g–j. g Sae2 and Dna2 display a cell cycle and DDK-dependent shift in electrophoretic mobility. Cells expressing 9Myc-tagged resection proteins as indicated were arrested either in G1 or M-phase and samples run on gels. Data are representative of n = 2–4 biological replicates. See also Supplementary Fig. 2k. Source data are provided as a Source Data file.