Fig. 4: MUS81-LIG4-PRIMPOL axis mediates replication restart following ROS-induced fork stalling.

a Western blot analysis of the extracts of U2OS cells transfected with indicated siRNAs. b ZRANB3 depletion restores replication fork progression in HU-treated U2OS cells in a manner dependent on PRIMPOL and the proteins required for restarting R-loop-stalled forks. Top: Experimental workflow. Bottom: Scatter plot of the values of IdU/CldU tract length ratio obtained from two independent experiments for indicated conditions (n ≥ 211). c, d Restart of DNA synthesis following replication arrest by HU depends on the same set of proteins as in b. c Experimental workflow of replication restart assays and representative images of replication tracts corresponding to restarted and stalled forks, respectively. Scale bar, 10 μm. d Quantification of the replication fork stalling events in cells depleted of indicated proteins. e–g MUS81 and PRIMPOL act in the same replication restart pathway. e Experimental workflow of replication restart assays with wild-type and MUS81 knockout (KO) HeLa Kyoto cells. f Western blot analysis of the extracts of cells in e transfected with indicated siRNAs. g Quantification of replication fork stalling events for the indicated conditions. h HU-induced fork stalling in MUS81-depleted U2OS cells depends on ROS. Top: Experimental workflow of replication restart assays. NAC, N-acetyl cysteine (5 mM). Bottom: Quantification of the replication fork stalling events for indicated conditions. d, g, h Data are presented as mean ± SD, n = 3; p values were calculated by one-way ANOVA followed by Tukey’s test. i Western blot analysis of the extracts of U2OS T-REx [RNH1(WT)-GFP] cells transfected with indicated siRNAs. j Restoration of replication fork progression in HU-treated cells by RNase H1 overexpression depends on MUS81 and PRIMPOL, but not RECQ1. Top: Experimental workflow of DNA fiber assays with U2OS T-REx [RNH1(WT)-GFP] cells. Doxycycline (Dox; 1 ng/ml) was added to induce RNase H1 (RNH1) expression. Bottom: Scatter plot of the values of IdU/CldU tract length ratio obtained from three independent experiments for indicated conditions (n ≥ 406). k Sensitivity of DNA replication tracts to S1 nuclease upon indicated conditions. Top: Experimental workflow of DNA fiber assays. TRP, triptolide (1 μM). Bottom: Scatter plot of the values of IdU/CldU tract length ratio obtained from three independent experiments for indicated conditions (n ≥ 417). b, j, k Red horizontal lines indicate the median; p values were calculated by Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Source data are provided as a Source Data file.