Fig. 2: Discn regulates DNA replication stress response and HR-mediated DNA DSB repair.

a Mouse ESCs were treated with hydroxyurea (HU) to induce replication stress. ATR-CHK1 kinase activation failed to be well sustained in two Discn knockdown cells (KD-1 and KD-2) when compared to the knockdown control (KD-C). Three independent experiments were repeated with similar results. b DNA fiber assay revealed that the stalled forks in Discn KD cells were prone to undergo degradation after HU treatment. 200 fibers from three independent replications were analyzed. c Discn KD compromised the stalled fork restart. At least 200 fibers from three independent replications were analyzed. d Discn KD enhanced dormant origin firing as indicated by the reduced mean fork spacing. At least 50 continuous fibers from three independent replications were analyzed. e Discn KD increased the content of ssDNA as measured by the native BrdU incorporation assay at S phase. Scale bar, 10 μm. Quantification is shown on the right. At least 20 images were analyzed in each condition. f Compared to control ESCs (KD-C), Discn KD cells were more sensitive to hydroxyurea treatment and accumulated more DNA DSBs as measured by neutral comet assay. Data were from three replications and shown as mean ± SEM. g FACS analysis of reporter ESCs showed that Discn KD suppressed HR-mediated DNA DSB repair, but inversely stimulated NHEJ repair pathway. Data were from three independent experiments. h DNA DSBs were generated by laser microirradiation. Discn KD impaired the recruitment of Rad51 to DSB sites labeled with γH2AX at S phase (BrdU⁺), indicating the suppression of HR pathway. Right panel showed the proportions of S phase cells capable of HR repair (three replicates, 50 cells in each replicate). Scale bar, 5 μm. i Immunoblotting showed a decrease in chromatin-bound Rad51 in Discn KD ESCs synchronized at S phase. Three independent experiments were repeated with similar results. b, c, f–h Data were shown as mean ± SEM, two-tailed Student’s t-test. d, e Data were representative of individual values with box and whiskers plots showing the median, upper and lower quartiles, and minimum and maximum. Two-tailed Student’s t-test.