Fig 2: BRCA2 suppresses PRIMPOL-mediated repriming and ssDNA gap formation after DNA damage.a Western blots showing siRNA-mediated depletion of HLTF, SMARCAL1, and ZRANB3 in U2OS cells. Two siRNAs for each gene were used. Similar results were obtained in n = 3 independent experiments. b, c Replication tract lengths in U2OS cells depleted of BRCA2, HLTF, SMARCAL1, or ZRANB3 either before (b) or 6 h after (c) 10 Gy of IR. d Replication tract lengths in U2OS cells co-depleted of BRCA2 with HLTF, SMARCAL1, or ZRANB3 at 6 h after 10 Gy of IR. e Western blots showing the levels of PRIMPOL depletion with two different siRNAs. Similar results were obtained in n = 3 independent experiments. f Replication tract lengths in U2OS cells depleted of PRIMPOL alone or co-depleted of PRIMPOL and BRCA2 before and 6 h after 10 Gy of IR. g Effect of S1 nuclease treatment on replication tract lengths in control and BRCA2-depleted U2OS cells at 6 h after 10 Gy of IR. h Replication tract lengths in U2OS cells depleted of BRCA2, HLTF, SMARCAL1, or ZRANB3 alone or in combination with PRIMPOL at 6 h after 10 Gy of IR. Data in (b–d) and (f–h) are presented as mean ± s.d., with the number of dots shown below each column. P values are calculated using two-tailed unpaired Student’s t test. Source data are provided as a Source Data file.

Fig 3: 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.

Fig 4: Fork Progression, Fork Remodeling and Chromosomal Integrity Defects upon Inactivation of Different ZRANB3 Domains(A) Left: schematic representation of ZRANB3 domain organization and of mutations analyzed in this study. Right: western blot analysis of ZRANB3 in the indicated cell lines.(B) The indicated stable cell lines, expressing WT or mutant ZRANB3, were used for DNA fiber analysis as in Figure 1A, upon optional CPT 50 nM treatment. At least one hundred tracts were scored per sample. Whiskers: 10th–90th percentile (****p < 0.0001; ns, non-significant; Mann-Whitney test). Similar results were obtained in at least two biological replicates.(C) Frequency of replication fork reversal in the indicated cell lines, assessed by EM visualization, upon optional 1 hr treatment with CPT 50 nM. In brackets, the number of analyzed molecules. Similar results were obtained in two biological replicates (Table S4).(D) Left: number of chromosomal abnormalities per indicated cell line, as determined by metaphase spreading upon optional 8 hr CPT treatment (50 nM) and 16 hr nocodazole treatment (200 ng/mL). Error bars, standard deviations. Right: representative DAPI stained metaphase; the arrow points to a chromosome break. Scale bar, 5 µm. Western blot analysis of ZRANB3 protein levels in U2OS cell lines used in (D, left). In (A) and (D), the expression level of HA-tagged ZRANB3 WT and mutant proteins (retarded mobility) is close to endogenous ZRANB3 levels in U2OS cells. ß tubulin, loading control.

Fig 5: RAD51 and HEXIM1 Modulate BET Inhibitor-Induced DNA Damage(A) Representative images of DNA damage foci after JQ1 treatment. HU was 2 mM 8 hr.(B) Representative images of RAD51 foci after JQ1 treatment.(C) Total percentages (top) and percentage normalized to S/G2 content (bottom) of cells with RAD51 foci after JQ1 treatment (n = 3–6).(D) Percentages of cells with RAD51 foci ± 4 hr JQ1 and a-amanitin (AM) (n = 3).(E) EU and DNA fiber labeling after RAD51 depletion.(F) Protein levels of RAD51 after siRNA depletion.(G) Replication fork speeds after 1 hr JQ1 ± RAD51 siRNA (n = 3 and 4).(H) Replication fork speeds after 1 hr JQ1 ± PARP inhibitor (n = 3).(I) Protein levels of SMARCAL1 or ZRANB3 48 hr after siRNA transfection.(J) Replication fork speeds after 4 hr JQ1 ± SMARCAL1 or ZRANB3 siRNA (n = 3).(K) Representative images of ?H2AX and 53BP1 foci after JQ1 ± RAD51 siRNA.(L) Percentages of cells with ?H2AX foci after 8 hr JQ1 ± RAD51 siRNA (n = 4).(M) Percentages of cells with 53BP1 foci after 8 hr JQ1 ± RAD51 siRNA (n = 4).(N) Percentages of cells with ?H2AX foci after JQ1 ± HEXIM1 siRNA (n = 3).(O) Percentages of cells with 53BP1 foci after JQ1 ± HEXIM1 siRNA (n = 3).(P) Model: BET inhibition disrupts recruitment of chromatin and replication factors; increased RNA synthesis and RAD51 activity (e.g., fork reversal) slow replication forks without DNA damage.Data are represented as mean ± SEM. Scale bars, 10 µm. See also Figure S4.