Fig. 2: BCDX2 paralog complex, but not CX3, promotes replication fork slowing and reversal upon mild CPT treatment.

a Rationale for focusing on RAD51C, RAD51D, and XRCC3 downregulation, to perform functional studies on BCDX2 and CX3 complexes during replication stress. Top: schematic of the two main RAD51 paralog complexes. Bottom: differential effects of RAD51C, RAD51D, and XRCC3 inactivation on paralog complex stability/function. b Western blot analysis of RAD51 paralogs upon 48 h downregulation with two different siRNAs. Ctrl, control siRNA; Tubulin, loading control. asterisk, specific band. In b and d multiple gels/blots were processed in parallel, ensuring equal and comparable loading across gels. The experiment was performed twice yielding similar results. c DNA fiber analysis of U2OS cells 48 h after transfection with a control siRNA (siCtrl) or with siRNAs targeting RAD51C, RAD51D, and XRCC3. Top-left: schematic of the CldU/IdU pulse-labeling protocol used to evaluate fork progression upon 50 nM CPT treatment. Bottom-left: representative DNA fibers from each genetic condition. Left: IdU/CIdU ratio is plotted as a readout of fork progression. In c and e, the numbers indicate the mean value; a minimum of 150 forks was scored in two independent experiments yielding similar results. Bounds of box are 25–75th percentile, center shows the median, whiskers indicate the 10–90 percentiles, data points outside this range are drawn as individual dots. Statistical analysis: Kruskal–Wallis test; ns not significant; ****p-value < 0.0001, ***p-value = 0.0003. d Western blot analysis of RAD51 paralogs in Knock-Out (KO) U2OS cells and in cells reconstituted of the respective protein (+). KU70, loading control. The experiment was performed twice yielding similar results. e DNA fiber analysis of KO and reconstituted (+) U2OS cells labeled as in c. f, g Frequency of reversed replication forks in U2OS cells transfected with control siRNA (Ctrl) or with siRNAs targeting RAD51C, RAD51D, or XRCC3 for 48 h and treated with 50 nM CPT (1 h). f Electron micrograph of a representative reversed replication fork from CPT-treated U2OS cells (P parental duplex, D daughter duplexes, R regressed arm; scale-bar, 100 nm). g Graph-bar showing mean and SD from three independent EM experiments. In brackets, total number of molecules analyzed per condition. Statistical analysis: one-way ANOVA followed by Bonferroni test; ns not significant; ***p-value = 0.0004. Source data for b–g are provided in the Source Data file.