Fig. 5: BCDX2, but not CX3, promotes chromosomal instability in BRCA2-defective cells.

a Metaphase spread analysis for detecting chromosomal aberrations in U2OS cells double transfected with a control siRNA (Ctrl) or with siRNAs targeting RAD51C, RAD51D, and XRCC3 for 60 h and with BRCA2 siRNA for 48 h and treated with 4 mM HU for 5 hr. Left: representative metaphase spread image (scale-bar, 5 μm); 1, 2, 3 representative breaks and 4 representative intact chromosome. Center: number of chromosomal abnormalities for each genetic condition. The graph-bar depicts mean and SD from at least three independent experiments. Statistical analysis: one-way ANOVA followed by Bonferroni test; ns not significant; **p-value = 0.0012 (5 versus 6) and 0.0020 (5 versus 7); ***p-value = 0.0001; ****p-value < 0.0001. Right: levels of indicated proteins, assessed by western blot; Tubulin, loading control; asterisk, specific band. Multiple gels/blots were processed in parallel, ensuring equal and comparable loading across gels. Source data are provided in the Source Data file. b Proposed model for the sequential role of BCDX2 and CX3 paralog complexes in replication fork remodeling, protection and restart: the BCDX2 complex helps RAD51 and ZRANB3 in driving fork remodeling. During fork stalling, regressed arms are protected from unscheduled nucleolytic degradation by BRCA2-mediated stabilization of RAD51 nucleofilament. Preventing fork remodeling by BCDX2 inactivation suppresses genetic instability of BRCA2-deficient cells upon sustained replicative stress. Similarly to RAD51, BCDX2 may be involved in fork protection (and possibly fork restart), but this role may be genetically masked by its upstream role in promoting fork reversal. Once DNA synthesis can resume, the CX3 complex promotes reversed fork restart, likely by engaging RAD51-bound regressed arms in efficient strand invasion events. In absence of fork remodeling, stalled forks undergo a fast, but possibly inaccurate restart mode, likely driven by repriming events.