Fig. 1: ROS-induced replication slowdown causes replication fork stalling.

a–e Elimination of ROS or PRDX2 inhibition prevents nascent DNA degradation in BRCA2-depleted U2OS cells upon exposure to hydroxyurea (HU). a Experimental workflow of DNA fiber assays. ConA, Conoidin A (PRDX2 inhibitor; 0.5 μM); NAC, N-acetyl cysteine, (ROS scavenger; 5 mM); NUC, exogenous nucleosides (adenosine, guanosine, thymidine and cytosine; 20 μM each), Mirin, (MRE11 inhibitor; 50 μM). A thymidine (400 μM) chase was included for all conditions to stop IdU incorporation. b Representative images of DNA replication tracts. Scale bar, 10 μm. c Western blot analysis of the extracts of U2OS cells transfected with control siRNA (siLuc) or siRNA to BRCA2 (siBRCA2). d Scatter plot of the values of IdU/CldU tract length ratio obtained from three independent experiments for indicated conditions (n ≥ 371). e Scatter plot of the values of sister IdU tract length ratio (sister fork ratio; shorter tract/longer tract) obtained from three independent experiments for indicated conditions (n ≥ 100). f–i ROS induce replication fork stalling in a PRDX2 dependent manner in U2OS cells. f Experimental workflow. ConA and NAC were present at concentrations as in (a). g Representative DNA fiber images. NT, non-treated. Scale bar, 10 μm. h Scatter plot of the values of IdU/CldU tract length ratio obtained from three independent experiments for indicated conditions (n ≥ 371). i Scatter plot of the values of sister fork ratio obtained from three independent experiments for indicated conditions (n ≥ 90). j–m ZRANB3 depletion rescues HU-induced fork stalling in U2OS cells. j Experimental workflow. k Western blot analysis of the extracts of U2OS cells transfected with indicated siRNAs. l Scatter plot of the values of IdU/CldU tract length ratio obtained from three independent experiments for indicated conditions (n ≥ 402). m Scatter plot of the values of sister fork ratio obtained from three independent experiments for indicated conditions (n ≥ 96). d, e, h, i, l, m 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.