Fig. 1: DDK phosphorylates HR proteins and is required for HR. | Nature Communications

Fig. 1: DDK phosphorylates HR proteins and is required for HR.

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

Fig. 1

a DDK mutants are hypersensitive to camptothecin (CPT). Five-fold serial dilutions of bob1-1, bob1-1 dbf4Δ, and bob1-1 cdc7Δ 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. 1a, b. 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 dbf4Δ cells. n = 4 biological replicates. See also Supplementary Fig. 1c–h. 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.

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