Fig. 3: DDK regulates short-range resection via phosphorylation of Sae2.
a A model CDK (mammalian CDK2-CycA) and budding yeast DDK can phosphorylate Sae2 in vitro. Top, autoradiography monitoring incorporation of radioactive phosphate; Bottom, same gel stained with Coomassie Blue. Note that DDK can auto-phosphorylate, as previously shown15,85. Data are representative of n = 2 independent experiments. b, c Sae2 (b) or Sae2-S267E (c) were phosphorylated by DDK or mock-treated, and added to the MRX complex to monitor endonucleolytic clipping of DNA. Left, quantification of cleavage products resolved on gels such as shown on the right. n = 3 independent experiments, shown is the mean with values of replicates, error bars denote SD. Reported p-values were calculated using a two-tailed unpaired t-test. d Inverted Alu repeats (Alu-IR) at the LYS2 locus induce Sae2-MRX endonucleolytic cleavage and recombination with a locus carrying a truncated version of LYS2 (lys2::Δ5’). e Recombination rates were calculated using a fluctuation analysis. n = 3, 7–8 fluctuations used per replicate per strain. Box plot shows mean with values of biological replicates, error bars denote SD. f ssDNA accumulation is monitored by resistance to restriction enzyme cleavage after DSB induction via the HO nuclease at the MAT locus. The exo1Δ sgs1-AID dna2-AID background make the assay specific for Sae2-MRX-dependent short-range resection. RS = restriction site. g, h Depletion of Dbf4 induces defect in Sae2-MRX mediated resection. ssDNA accumulation upon DSB induction measured 98 bp downstream (g) and 120 bp upstream (h) the DSB via qPCR after digestion with restriction nucleases RsaI and MseI, respectively. n = 6 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. See also Supplementary Fig. 3c, d. i Scheme of Sae2 highlighting S/T-D/E sites and S267. j 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. 3i. k, l Five-fold serial dilutions of indicated strains were grown on YPD plates or YPD plates supplemented with CPT at the indicated concentrations. Data are representative of n = 3 biological replicates. See also Supplementary Fig. 3i, l. Source data are provided as a Source Data file.
