Fig 2: Genetic interaction between PCNA ubiquitination and the BRCA pathway.a DNA fiber combing assay showing that concomitant loss of BRCA2 and PCNA ubiquitination enhances nascent strand degradation upon HU-induced fork arrest. The ratio of CldU to IdU tract lengths is presented, with the median values marked on the graph and listed at the top. Asterisks indicate statistical significance (Mann–Whitney test, two-sided). A schematic representation of the fiber combing assay conditions is also presented. b Immunofluorescence experiment showing synergistic increase in 53BP1 foci formation in unsynchronized K164R cells upon BRCA2 depletion. At least 100 cells were quantified for each condition (pooled from two experiments). The mean values are marked on the graph, and asterisks indicate statistical significance (t-test, two-tailed, unequal variance). Representative micrographs are also shown. c BrdU-alkaline comet assay measuring ssDNA gaps under normal replication conditions in BRCA2-depleted 293T cells at 1 and 5 h after BrdU pulse. At least 100 cells were quantified for each condition. Center line indicates the median, bounds of box indicate the first and third quartile, and whiskers indicate the 10th and 90th percentile. Asterisks indicate statistical significance (t-test, two-tailed, unequal variance). A schematic representation of the assay conditions is also presented. d, e Clonogenic survival experiments showing that loss of PCNA ubiquitination does not result in olaparib sensitivity, but it drastically increases the olaparib sensitivity of BRCA1-depleted d and BRCA2-depleted e cells. The average of three experiments, with standard deviations indicated as error bars, is shown. Asterisks indicate statistical significance (t-test, two-tailed, unequal variance) comparing WT siBRCA1/2 to KR siBRCA1/2. Representative images of the clonogenic assays are presented in Supplementary Fig. 8c, d. f, g Clonogenic survival experiments showing that depletion of RAD18 f or ATAD5 g increases the olaparib sensitivity of BRCA2-knockout HeLa cells. The average of three experiments, with standard deviations indicated as error bars, is shown. Asterisks indicate statistical significance (t-test, two-tailed, unequal variance) comparing BRCA2KO siControl to BRCA2KO siRAD18/siATAD5. Source data are provided as a Source Data file.

Fig 3: Loss of CCNC is important for survival and PARPi sensitivity in BRCA2-deficient Cells. (A) Validation of CCNC KO in HEK293A BRCA2 mAID-EGFP KI cells by western blot using the indicated antibodies. (B) Results of long-term clonogenic assays conducted using HEK293A BRCA2 mAID-EGFP CCNC-WT and -KO cells with the indicated treatments. Ola: Olaparib. (C) Quantification of relative cell survival in the clonogenic assays in (B). All treated groups were normalized to the indicated untreated group. The data are presented as means ± SD. ***P < 0.001 (Student's t-test). (D) Results of long-term clonogenic assays conducted using HEK293A BRCA2 mAID-EGFP CCNC-WT and -KO cells with the indicated treatments. Tal: Talazoparib. (E) Quantification of relative cell survival in the clonogenic assays in (D). All treated groups were normalized to the indicated untreated group. The data are presented as means ± SD. ***P < 0.001 (Student's t-test). (F) Capan-1 cells were infected with control pLentiCRISPRv2 (Lenti V2), or CCNC gRNA-1 (g1) virus and selected with puromycin. Western blot was conducted to validate CCNC knock down efficiency with the indicated antibodies. (G) Results of long-term clonogenic assays conducted using Capan-1 LentiV2 and CCNC g1 cells with different concentration of Olaparib treatments. Ola: Olaparib. (H) Quantification of relative cell survival in clonogenic assays in (G). All treated groups were normalized to the indicated untreated group. The data are presented as means ± SD. ***P < 0.001 (Student's t-test). (I) Results of long-term clonogenic assays conducted using Capan-1 LentiV2 and CCNC g1 cells with different concentration of Talazoparib treatments. Tal: Talazoparib. (J) Quantification of relative cell survival in the clonogenic assays in (I). All treated groups were normalized to the indicated untreated group. The data are presented as means ± SD. **P < 0.01, ***P < 0.001 (Student's t-test).

Fig 4: E2F7 depletion restores RAD51-mediated HR in BRCA2-deficient cells. (A) DR-GFP assay in U2OS cells showing that E2F7 depletion restores HR levels in BRCA2-knockdown cells, but not in RAD51-knockdown cells. The average of three to five experiments, with standard deviations as error bars, is shown. Asterisks indicate statistical significance. (B) EJ5-GFP assay showing the impact of E2F7 co-depletion on the NHEJ levels in BRCA2-depleted cells. The average of four to six experiments, with standard deviations as error bars, is shown. Asterisks indicate statistical significance.

Fig 5: CDC7 contributes to replication‐dependent chromosomal breakagesU2OS or AS‐CDC7 cells were transfected with control (siCon) or BRCA2 (siBRCA2)‐targeting siRNAs and then either mock‐treated or treated with 4 mM HU for 5 h in the presence or absence of 10 μM XL413 or 3MB‐PP1, respectively. HU was then washed off and nocodazole was added to the medium in order to complete one round of DNA synthesis. Metaphase spreads were prepared and analysed. ARepresentative metaphase spread from BRCA2‐depleted U2OS cells treated with HU. Scale bar = 5 μm. Arrows indicate the chromosomes enlarged in the insets, two of which show chromatid breaks (*).B, CThe graphs show the average number of chromatid breaks per spread. In each experiment, 30 chromosome spreads for each condition were analysed and three independent experiments were performed. Error bars represent SEM. Statistical significance was assessed by Student's t‐test (**P ˂ 0.01, ***P < 0.001).D, EU2OS and AS‐CDC7 cells were transfected with control (siCon) or BRCA2 (siBRCA2)‐targeting siRNAs. Forty‐eight hours post‐transfection, cells were either mock‐treated or treated with 10 μM XL413 or 10 μM 3MB‐PP1, respectively, for 24 h with nocodazole added for the last 16 h of the experiment. The graph shows the average number of chromatid breaks per spread. In each experiment, 30 chromosome spreads for each condition were analysed and three independent experiments were performed. Error bars represent SEM. Statistical significance was assessed by Student's t‐test (*P < 0.05, ***P < 0.001). Source data are available online for this figure.