Dbf4-dependent kinase promotes cell cycle controlled resection of DNA double-strand breaks and repair by homologous recombination

DNA double-strand breaks (DSBs) can be repaired by several pathways. In eukaryotes, DSB repair pathway choice occurs at the level of DNA end resection and is controlled by the cell cycle. Upon cell cycle-dependent activation, cyclin-dependent kinases (CDKs) phosphorylate resection proteins and thereby stimulate end resection and repair by homologous recombination (HR). However, inability of CDK phospho-mimetic mutants to bypass this cell cycle regulation, suggests that additional cell cycle regulators may be important. Here, we identify Dbf4-dependent kinase (DDK) as a second major cell cycle regulator of DNA end resection. Using inducible genetic and chemical inhibition of DDK in budding yeast and human cells, we show that end resection and HR require activation by DDK. Mechanistically, DDK phosphorylates at least two resection nucleases in budding yeast: the Mre11 activator Sae2, which promotes resection initiation, as well as the Dna2 nuclease, which promotes resection elongation. Notably, synthetic activation of DDK allows limited resection and HR in G1 cells, suggesting that DDK is a key component of DSB repair pathway selection.

filtered for GOBP:double strand break repair  (d) Pipeline for the analysis of the phospho-proteome (and total proteome) using Perseus (v1.6.5.0).(e-g) Heat maps depicting the z-score, highlighting phospho-peptides from the DDK cluster after filtering in Perseus (v1.6.5.0) for (e) GOBP: double-strand break repair (GO: 0006302), (f) GOBP: DNA damage response (GO:0006974) and (g) GOBP: chromatin remodeling (GO:0006338).Gene name and modified residues are reported.GOBP = Gene Ontology Biological Process.Related to experiment shown in Fig. 1d.(h) Heat map depicting the z-score, highlighting the Xrs2 phospho-peptide identified in the DDK cluster.Related to experiment shown in Fig. 1d.Source data are provided as a Source Data file.(i) Western blot to monitor total protein levels of Sae2, Sae2-6A and Sae2-14A from sae2D strains complemented with ectopically expressed Sae2 variants 9Myc tagged.Pgk1 is used as a loading control.Sae2-9Myc and the 6A mutant appear to have similar levels, while the 14A mutant appear to have slightly reduced total protein levels.
(j) DNA content measurement by flow cytometry confirmed G1 or M-phase cell cycle arrest of cells expressing Sae2-9Myc variants.Related to experiment shown in Supplementary Fig. 3k.'async.' stands for asynchronous.
(k) Phospho-shift of Sae2-6A and Sae2-14A mutants.Cells of indicated strains were arrested in G1 or M-phase to monitor the Sae2 phospho-shift.Data are representative of n=3 biological replicates.See also Supplementary Fig. 3j.
(l) Western blot to monitor total protein levels of Sae2, Sae2-6A, Sae2-S267A and Sae2-7A (S267A + 6A) from sae2D strains complemented with ectopically expressed Sae2 variants 9Myc tagged.Pgk1 is used as a loading control.Source data are provided as a Source Data file.(b) Evidence for identified phospho-peptides carrying serine 236 and serine 237 of Dna2.Serine 236 is highlighted in orange while serine 237 is highlighted in green.Double-phosphorylated peptides (S236-phospho/S237-phospho) are identified in all replicates of WT and bob1-1 controls (with probability of 100% for both serine).In contrast, only singly phosphorylated peptides are identified in all replicates of the bob1-1dbf4D strain, with the phosphorylation always assigned with high probability (80% up to 95%) to serine 237, suggesting that DDK is required specifically for the phosphorylation of serine 236.

DSB (h)
phospho-peptides from proteins found to be significantly different in both total proteome analysys 7-hierarchical clustering (heat map Figure1d) 8 DNA content measurement by flow cytometry to confirm M-phase cell cycle arrest.Data are representative of n=3 biological replicates.Related to experiment shown in Fig. 1c.'async.' stands for asynchronous.(b) DDK mutants are defective in survival by HR.Using system from Fig. 1b (to monitor HR-dependent cell survival), five-fold serial dilutions of the reported strains (with or without the donor template) were spotted on YPD (as control) or YPGal (for chronic pGAL-HO-induction) plates.Data are representative of n=3 biological replicates.(c) DNA content measurement by flow cytometry to confirm M-phase cell cycle arrest.Related to experiment shown in Fig. 1d.'async.' stands for asynchronous.
Enrichment of RPA in bob1-1 and bob1-1cdc7D cells, monitored via ChIP-qPCR at an HO-induced DSB at the MAT locus using several primer pairs (up to 20 kb downstream the DSB).Experiments were performed using cells arrested in M-phase.n=2, values of biological replicates are shown; curves display the mean values.(b-c) Controls to test the Dbf4-3AID system.(b) M-phase arrested dbf4-3AID cells were either mock treated or treated with 1 mM IAA. (c) Five-fold serial dilutions of WT or dbf4-3AID cells were spotted on YPD plates (as control) or YPD plates supplemented with 1 mM NAA.(d) Same as in (a), but using dbf4-3AID cells mock or treated with 1 mM IAA prior DSB induction.(e-f) (e) Western blot with anti-AID and an anti-Dbf4 antibodies to control Dbf4-3AID degradation in IAA-treated cells.(f) DNA content measurement by flow cytometry to confirm M-phase arrest.'async.' stands for asynchronous.Data are representative of n=4 biological replicates.Related to experiment shown in Fig. 2d-e.(g) DNA content measurement by flow cytometry to confirm M-phase cell cycle arrest.Data are representative of n=2 biological replicates.Related to experiment shown in Fig. 2f.'async.' stands for asynchronous.(h) Western blot of samples to monitor DDK-inhibition using MCM2-phosphoS53 (MCM2-P) as a proxy for DDK activity and gH2AX as a proxy for DNA damage induction.Data are representative of n=2 biological replicates.Related to experiment shown in Fig. 2f.(i-j) (i) Quantification of the MCM2-phosphoS53 (MCM2-P) signal relative to GAPDH as loading control.(j) Quantification of the gH2AX signal relative to GAPDH as loading control.n=2, box plot shows mean with values of biological replicates.Related to experiment shown in Fig. 2f.(k) DNA content measurement by flow cytometry to confirm G1 or M-phase cell cycle arrest of the reported strains.Data are representative of n=2-4 biological replicates.Related to experiment shown in Fig. 2g.(l) Sae2 and Dna2 gel-shift is sensitive to l-phosphatase.Soluble extracts from M-phase cells expressing Sae2-9Myc or Dna2-9Myc (input) were split to be mock or l-phosphatase-treated and the gel-shift monitored on gel.Data are representative of n=2 technical replicates.(m-p) cdc28-as1 cells carrying Sae2-9Myc (m-n) or Dna2-9Myc (o-p) were arrested in G1 or M-phase and either mock treated or treated with 1.5 µM 1-NM-PP1 to inhibit CDK.Upon CDK inhibition, (n) the Sae2 phospho-shift is reduced, while (p) the Dna2 phospho-shift is largely abolished.Sld2-CDKdependent phospho-shift was used as a control for CDK inhibition.DNA content measurement by flow cytometry confirmed G1 or M-phase cell cycle arrest of cells expressing Sae2-9Myc (m) or Dna2-9Myc (o).Data are representative of n=3 biological replicates.'async.' stands for asynchronous.Source data are provided as a Source Data file.
cl.2 cl.1 cl.2 cl.1 cl.2 cl.1 cl.2 cl.1 cl.2 cl.1 cl.2 S/T-D/E to A S/T-D/E + S/T-S/T to of the clipping assay used to monitor endonucleolytic clipping by Sae2-MRX.32P-labelled linear DNA with streptavidin blocked ends was used as a substrate of Sae2-MRX.Endonucleolytic DNA cleavage was monitored via the appearance of cleaved DNA products after denaturing electrophoresis.Sae2 was purified separately from MRX, allowing to pre-phosphorylate (or mock-treat) Sae2 before reconstituting the Sae2-MRX complex and monitor its endonucleolytic activity.(b) Sae2-MRX endonucleolytic activity is slightly higher after CDK phosphorylation of Sae2 compared to DDK phosphorylation.Sae2 was phosphorylated by CDK, DDK or mock treated and added to the MRX complex.Sae2-MRX mediated endonucleolytic clipping of DNA was then monitored on denaturing gels.Top, quantification of the DNA cleavage products; bottom, a representative experiment, n=4, box plot depicts mean with values of replicates, error bars denote SD.Reported p values were calculated using a two-tailed unpaired t-test.(c) Western blot to confirm Dbf4-3AID degradation after treatment with IAA in the strains used for the experiment shown in Fig.3g-h.Cells were arrested in M-phase and treated with 1 mM IAA.Samples were then loaded on a gel to monitor Dbf4-3AID.Note that Dna2-AID and Sgs1-AID run at the same height and are both degraded after IAA treatment as well.(d) Cutting efficiency of the HO nuclease at the MAT locus measured via qPCR.The cut efficiency is used to normalize the amount of ssDNA to the amount of cut DNA.n=6 biological replicates, shown is mean with values of replicates, error bars denote SD.Related to the experiment shown in Fig.3g-h.(e-g) Sae2-MRX mediated short-range resection is reduced upon depletion of Dbf4 and largely abolished in sae2-S267A mutants.ssDNA accumulation upon DSB induction measured at sites 98 bp downstream (e) and 120 bp upstream (f) the DSB via qPCR after digestion with restriction nucleases RsaI and MseI, respectively.(g) Cutting efficiency of the HO nuclease at the MAT locus measured via qPCR.The cut efficiency is used to normalize the amount of ssDNA to the amount of cut DNA.n=6 (WT and dbf4-3AID), n=3 (sae2-S267A and sae2-S267A dbf4-3AID) biological replicates.Shown is mean with values of replicates, error bars denote SD.Reported p values were calculated using a two-tailed unpaired t-test.The data for WT and dbf4-3AID in Supplementary Fig.3e, Supplementary Fig.3fand Supplementary Fig.3gare the same as in Fig.3g, Fig.3hand Supplementary Fig.3d, respectively.(h) Scheme of Sae2 highlighting S/T-D/E and S/T-S/T sites.
Highlight of serine 236 and serine 237 of Dna2.Data from phosphoproteomic experiment of Fig.1d.Heat-map depicts the z-score.
(c) Western blot to monitor total protein levels of Dna2-9Myc and Dna2-S236A-9Myc.Pgk1 is used as a loading control.Dna2-9Myc and Dna2-S236A-9Myc have similar total protein levels.(d) ) DNA content measurement by flow cytometry confirmed G1 or M-phase cell cycle arrest of cells expressing Dna2-9Myc variants.Related to experiment shown in Fig. 4c.'async.' stands for asynchronous.(e) Serine 236 mutation to alanine affects the Dna2 phosphorylation shift to an extent similar to the lack of DDK.Cells of the indicated strains were arrested in G1 or M-phase and samples collected and loaded on a gel to monitor the Dna2 phospho-shift.Data are representative of n=2 biological replicates.(f) Western blot to confirm Dbf4-3AID degradation after treatment with IAA in the strains used for experiments shown in Fig. 4e-f.Cells were arrested in Mphase and treated with 1 mM IAA.Samples were then loaded on gel to monitor Dbf4-3AID.(g-h) Cutting efficiency of the HO nuclease at the MAT locus measured via qPCR.The cut efficiency is used to normalize the amount of ssDNA to the amount of cut DNA.n=3 biological replicates, shown is mean with values of replicates, error bars denote SD.(g) Related to the experiment shown in Fig. 4e.(h) Related to the experiment shown in Fig. 4f.Source data are provided as a Source Data file.