Journal: Nature Communications

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

doi: 10.1038/s41467-024-46951-z

Figure Lengend Snippet: a DDK mutants are hypersensitive to camptothecin (CPT). Five-fold serial dilutions of bob1-1 , bob1-1 dbf4 Δ, and bob1-1 cdc7 Δ strains were spotted on YPD or YPD containing CPT at the indicated concentrations. Data are representative of n = 2 biological replicates. b Scheme of the gene conversion assay used to study HR rates. A DSB generated by the HO endonuclease at 491 kb (Chr. IV) can be repaired using a homologous donor template at 795 kb (Chr. IV). The latter carries a unique 23-bp sequence, allowing for quantification of the recombination products via qPCR; arrowheads denote PCR primer locations. Gene conversion using the donor template disrupts the HO cut site, allowing cell survival to the chronic induction of the endonuclease as an alternative readout. ‘cfu’ stands for colony-forming unit. c DDK is required for HR. qPCR analysis of HR using system as in ( b ) in cells arrested in M-phase. Cells lacking the donor template are used as control. 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. . d Class-I peptides derived from phospho-proteomic experiment were subjected to analysis of variance (ANOVA) test, with permutation-based false discovery rate (FDR) cutoff of 0.05. ANOVA significant phospho-peptides were then subjected to a hierarchical clustering in Perseus (v1.6.5.0). The calculated z-scores are shown in the heat-map and the different clusters are highlighted. The DDK cluster shows specific phospho-peptides downregulated in bob1-1 dbf4 Δ cells. n = 4 biological replicates. See also Supplementary Fig. . e Motif sequence generated for phospho-peptides enriched in the DDK cluster showing the 3 positions upstream and downstream the modified S/T. f Heat-map depicting the z-score, highlighting phospho-peptides from the DDK cluster after filtering in Perseus (v1.6.5.0) for GOBP:DNA repair (GO: 0006281). Gene name and modified residues are reported. GOBP = Gene Ontology Biological Process. Source data are provided as a Source Data file.

Article Snippet: Reported p -values were calculated using a two-tailed unpaired t -test with the Graphpad t -test calculator web tool ( https://www.graphpad.com/quickcalcs/ttest1.cfm ; Graphpad), with exception for the p- values reported in Fig. , Fig. , Supplementary Fig. which were calculated using a two-tailed unpaired t -test with R and the p -values reported in Fig. which were calculated using a two-tailed Mann-Whitney test (Prism 8, Graphpad). p -values reported in Supplementary Data were calculated selecting Fisher’s exact test in the GOBP analysis with Panther ( https://geneontology.org ).

Techniques: Generated, Sequencing, Control, Two Tailed Test, Derivative Assay, Modification

Journal: Nature Communications

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

doi: 10.1038/s41467-024-46951-z

Figure Lengend Snippet: 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 shown , . 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:: Δ 5’ ). 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 exo1 Δ 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. . 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. . 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. , l. Source data are provided as a Source Data file.

Article Snippet: Reported p -values were calculated using a two-tailed unpaired t -test with the Graphpad t -test calculator web tool ( https://www.graphpad.com/quickcalcs/ttest1.cfm ; Graphpad), with exception for the p- values reported in Fig. , Fig. , Supplementary Fig. which were calculated using a two-tailed unpaired t -test with R and the p -values reported in Fig. which were calculated using a two-tailed Mann-Whitney test (Prism 8, Graphpad). p -values reported in Supplementary Data were calculated selecting Fisher’s exact test in the GOBP analysis with Panther ( https://geneontology.org ).

Techniques: In Vitro, Autoradiography, Staining, Two Tailed Test

Journal: Nature Communications

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

doi: 10.1038/s41467-024-46951-z

Figure Lengend Snippet: a Serine 236 of Dna2 is phosphorylated in a DDK-dependent manner. Data from phospho-proteomic experiment of Fig. . Heat-map depicts the z-score. See also Supplementary Fig. . b Scheme of Dna2 phosphorylation sites. Green: CDK target sites previously identified ; Orange: DDK target site S236, within S/T-S/T motif. c Serine 236 phosphorylation contributes to DDK-dependent Dna2 phosphorylation shift in vivo. 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 = 3 biological replicates. See also Supplementary Fig. . d dna2-S236A strain is sensitive to CPT, when EXO1 is deleted. Five-fold serial dilutions of WT, exo1 Δ , dna2-S236A, and dna2-S236A exo1 Δ strains were grown on YPD plates or YPD plates supplemented with CPT at the indicated concentrations. Data are representative of n = 3 biological replicates. e Depletion of Dbf4 induces defects in STR-Dna2-mediated long-range resection. ssDNA accumulation upon DSB induction was measured via qPCR after digestion with the RsaI restriction nuclease at sites with indicated distances from the DSB. n = 3 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. . f DDK-dependent resection defect accumulates in the absence of EXO1 . Resection was measured as in ( e ), but in a background where resection was carried out by Sae2-MRX and STR-Dna2. n = 3 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. f, . Source data are provided as a Source Data file.

Article Snippet: Reported p -values were calculated using a two-tailed unpaired t -test with the Graphpad t -test calculator web tool ( https://www.graphpad.com/quickcalcs/ttest1.cfm ; Graphpad), with exception for the p- values reported in Fig. , Fig. , Supplementary Fig. which were calculated using a two-tailed unpaired t -test with R and the p -values reported in Fig. which were calculated using a two-tailed Mann-Whitney test (Prism 8, Graphpad). p -values reported in Supplementary Data were calculated selecting Fisher’s exact test in the GOBP analysis with Panther ( https://geneontology.org ).

Techniques: Phospho-proteomics, In Vivo, Two Tailed Test

Journal: Nature Communications

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

doi: 10.1038/s41467-024-46951-z

Figure Lengend Snippet: 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 Dbf4 , . 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. . 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. . 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, yku80 Δ, and GAL-DDK yku80 Δ 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. . 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.

Article Snippet: Reported p -values were calculated using a two-tailed unpaired t -test with the Graphpad t -test calculator web tool ( https://www.graphpad.com/quickcalcs/ttest1.cfm ; Graphpad), with exception for the p- values reported in Fig. , Fig. , Supplementary Fig. which were calculated using a two-tailed unpaired t -test with R and the p -values reported in Fig. which were calculated using a two-tailed Mann-Whitney test (Prism 8, Graphpad). p -values reported in Supplementary Data were calculated selecting Fisher’s exact test in the GOBP analysis with Panther ( https://geneontology.org ).

Techniques: Activation Assay, Expressing, Phospho-proteomics, Negative Control, Comparison, Two Tailed Test, Control