Fig. 5: Synthetic activation of DDK activates limited DNA end resection and HR in G1 cells.
a Synthetic activation of DDK in G1-arrested cells. Codon-optimized versions of DBF4 and CDC7 are expressed from bidirectional pGAL1-10 promoter, with Dbf4 carrying D-box mutations that stabilize Dbf486,88. b Expression of DDK induces Sae2 phosphorylation in G1. Cells of the indicated strains were arrested in G1, DDK expression was induced by addition of galactose, and samples collected and loaded on a gel to monitor the Sae2 phospho-shift. Data are representative of n = 2 biological replicates. See also Supplementary Fig. 5a. c Synthetic activation of DDK in G1 allows for limited activation of DNA end resection. Strand-specific accumulation of 3′-ssDNA enriched by RPA-ChIP in G1-arrested cells indicates resection throughout time course (2, 4 h after DSB induction) in WT, sae2-S267E, GAL-DDK, and GAL-DDK sae2-S267E strains. RPA-ChIP signals at the DSB are normalized to DSB-independent RPA signals occurring throughout the genome. Data are representative of n = 2 biological replicates. See also Supplementary Fig. 5b-d. d, e Synthetic activation of DDK allows for limited recombination-mediated repair in G1. qPCR analysis of HR upon DSB induction at 491 kb (Chr. IV) using a donor template at 795 kb (Chr. IV). WT cells lacking the donor template are used as negative control. d Comparison of WT, sae2-S267E, GAL-DDK and GAL-DDK sae2-S267E strains. e Comparison of WT, GAL-DDK, yku80Δ, and GAL-DDK yku80Δ strains. n = 3, box plot shows mean with values of biological replicates, error bars denote SD. Reported p-values were calculated using a two-tailed unpaired t-test. See also Supplementary Fig. 5e-i. f Double kinase mechanism for cell cycle-regulated DNA end resection. CDK and DDK target at least two proteins, Sae2 to control Sae2-MRX-dependent short-range resection and Dna2 to control STR-Dna2-dependent long-range resection. Source data are provided as a Source Data file.
