Extended Data Fig. 8: Confirmation of DDK function in the regulation of DNA replication by H4S47 O-GlcNAcylation.
From: H4S47 O-GlcNAcylation regulates the activation of mammalian replication origins
a, Analysis of CDK2 and Cyclin E association with H4. HEK293T cells were transfected and treated as in Fig. 1a. IgG served as a negative control. b, c, Quantification of DBF4 and CDC7. WB from Fig. 4b (b) and Fig. 4c (c) were quantified, respectively. n = 3 biologically independent experiments. d, In vitro assay of H4 O-GlcNAcylation. His-H4WT and -H4S47A isolated from BL21 competent E. coli, which co-expressed OGT, were analyzed by WB with O-GlcNAcylation antibody CTD110.6. e, Quantification of DBF4, CDC7 and MCM2 pS53 levels for experiments in Fig. 4e. n = 3 biologically independent experiments. f, Overexpression of DBF4 enhances DNA replication. GFP-DBF4 or pEGFP-N1 (empty vector) was transfected along with Flag-H4WT or -H4S47A into HEK293T cells for 48 h prior to the detection of replication efficiency. From left, n = 4,183, 4,882, 3,133, 6,626 cells. P values were calculated by unpaired, two-tailed t-tests. For all box plots, the bottom, middle line and top of the box and whiskers indicate the 25th, 50th, 75th and 10–90th percentiles, respectively and means were shown as red ‘+’ signs. g, Quantification of MCM2/7 and DBF4 for experiments in Fig. 5c. n = 3 biologically independent experiments. h, DNA fiber assay. Experimental setup (left) and frequency distribution of inter-origin distances (right) for Fig. 6a were shown. i, Detection of H4S47 O-GlcNAcylation by mass spectrometry. Tandem mass spectrum of peptides containing S47 O-GlcNAcylation from experiments described in Fig. 6d was shown. The y, a and b fragmentations were used to map the O-GlcNAcylation site. P values calculated by Tukey’s multiple comparison test (two-way ANOVA), means ± s.d., n.s., non-significant (b, c, e and g). a.u., arbitrary unit.
