Fig 2: Differential regulation of nascent strand degradation by EXO1 and MRE11 by the KU complex.a, b PARP14-KU80 PLA experiments in HeLa cells showing that the interaction between PARP14 and KU is increased by BRCA2 deficiency. Representative micrographs, with scale bars representing 10 µm (a) and quantifications (b) are shown. The specificity of the readout is demonstrated by the loss of PARP14-KU80 PLA foci in HeLa-PARP14KO cells (a). At least 100 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard error of the mean, and asterisks indicate statistical significance (t-test, two-tailed, unpaired). c, d KU SIRF experiments showing that KU70 (c) and KU80 (d) binding to nascent DNA is increased by HU treatment and BRCA2 deficiency. At least 100 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard error of the mean, and asterisks indicate statistical significance (t-test, two-tailed, unpaired). Schematic representations of the assay conditions are shown at the top. Validation of the KU SIRF readout is shown in Supplementary Fig. 6b–d. e, f KU SIRF experiments showing that KU70 (e) and KU80 (f) binding to nascent DNA upon HU treatment in HeLa-BRCA2KO cells is suppressed by knockdown of fork reversal factors ZRANB3, SMARCAL1 and PARP1. At least 100 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard error of the mean, and asterisks indicate statistical significance (t-test, two-tailed, unpaired). Schematic representations of the assay conditions are shown at the top. Western blots confirming the depletions are shown in Supplementary Fig. 6e. g, h DNA fiber combing assays showing that knockdown of KU70 (g) or KU80 (h), with two different siRNA oligonucleotides for each, does not impact HU-induced fork degradation in HeLa-BRCA2KO cells. Western blots confirming KU70 and KU80 depletion are shown in Supplementary Fig. 6f. i DNA fiber combing assays showing that KU80 co-depletion restores fork degradation in PARP14-depleted HeLa-BRCA2KO cells. Western blots showing co-depletion of PARP14 and KU80 are presented in Supplementary Fig. 6g. j DNA fiber combing assays showing that KU80 depletion restores fork degradation in HeLa-BRCA2KOPARP14KO double knockout cells, and this degradation is not performed by MRE11 since it is not rescued by mirin treatment. k, l DNA fiber combing assays showing that KU80 co-depletion restores fork degradation in PARP14-depleted BRCA2-knockout cells, and this degradation is not performed by MRE11 since it is not rescued by mirin treatment. Co-depletion of EXO1, but not inhibition of MRE11 by mirin, restores fork protection in KU80-depleted BRCA2-knockout cells. Similar results were obtained in HeLa-BRCA2KO (k) and DLD1-BRCA2KO (l) cells. Western blots showing co-depletion of EXO1 and KU80 are presented in Supplementary Fig. 6h. m DNA fiber combing assays showing that EXO1 knockout restores fork protection in KU80-depleted HeLa-BRCA2KO cells. Two independent BRCA2KOEXO1KO double knockout clones were analyzed. Western blots confirming the EXO1 and BRCA2 knockout are shown in Supplementary Fig. 6i. For panels g–m, the ratio of CldU to IdU tract lengths is presented, with the median values marked on the graphs and listed at the top. At least 100 tracts were quantified for each sample. Asterisks indicate statistical significance (Mann-Whitney, two-tailed). Schematic representations of the assay conditions are shown at the top. Source data are provided as a Source Data file.

Fig 3: Regulation of EXO1 and MRE11 recruitment to nascent strand ssDNA gaps.a, b BrdU alkaline comet assays showing that treatment of EXO1-depleted HeLa-BRCA2KO cells with mirin does not further suppress the accumulation of ssDNA gaps induced by HU (a) or BPA (b) exposure. At least 40 nuclei were quantified for each condition. The median values are marked on the graph. Asterisks indicate statistical significance (Mann–Whitney, two-tailed). c, d PLA assays showing that EXO1 and MRE11 co-localize upon treatment with 0.4 mM HU (c) or 200 µM BPA (d) for 3 h. EXO1 deletion is used as control to confirm the specificity of the PLA signals observed. At least 75 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance (t test, two-tailed, unpaired). e, f EXO1 (e) and MRE11 (f) SIRF experiments showing the differential impact of the loss of each of these nucleases on the recruitment to nascent DNA of the other nuclease, upon exposure of HeLa-BRCA2KO cells to 0.4 mM HU. Loss of MRE11 suppressed EXO1 binding to nascent DNA, while EXO1 depletion did not affect MRE11 recruitment under these conditions. Depletion of MRE11 or EXO1 respectively is used as control to confirm the specificity of the SIRF signals observed. At least 45 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance (t test, two-tailed, unpaired). Schematic representations of the assay conditions are shown at the top. Source data are provided as a Source data file.

Fig 4: Loss of EXO1 suppresses the accumulation of nascent strand ssDNA gaps induced by replication stress in BRCA-deficient cells.a Schematic representation of the ssDNA gap expansion model tested: MRE11 extends the gap from the 3’ end and EXO1 extends it from the 5’ end. b, c BrdU alkaline comet assay showing that EXO1 knockdown suppresses the accumulation of replication-associated ssDNA gaps induced by treatment with 0.4 mM HU in HeLa-BRCA2KO (b) and RPE1-BRCA1KO (c) cells, similar to MRE11 depletion. At least 50 nuclei were quantified for each condition. The median values are marked on the graph and listed at the top. Asterisks indicate statistical significance (Mann–Whitney, two-tailed). A schematic representation of the assay conditions is shown at the top. Western blots confirming the EXO1 and MRE11 knockdown are shown in Supplementary Fig. S1a, b. d BrdU alkaline comet assay showing that EXO1 knockdown suppresses the accumulation of replication-associated ssDNA gaps induced by treatment with 150 µM cisplatin in HeLa-BRCA2KO cells, similar to MRE11 depletion. At least 75 nuclei were quantified for each condition. The median values are marked on the graph and listed at the top. Asterisks indicate statistical significance (Mann–Whitney, two-tailed). A schematic representation of the assay conditions is shown at the top. e, f S1 nuclease DNA fiber combing assays showing that knockdown of EXO1 suppresses the accumulation of nascent strand ssDNA gaps induced by treatment with 0.4 mM HU in HeLa-BRCA2KO (e) and RPE1-BRCA1KO (f) cells, similar to MRE11 depletion. The ratio of CldU to IdU tract lengths is presented, with the median values marked on the graphs and listed at the top. At least 45 tracts were quantified for each sample. Asterisks indicate statistical significance (Mann–Whitney, two-tailed). Schematic representations of the assay conditions are shown at the top. g S1 nuclease DNA fiber combing assays showing that knockdown of ZRANB3 or of SMARCAL1 does not affect the accumulation of nascent strand ssDNA gaps induced by treatment with 0.4 mM HU in HeLa-BRCA2KO cells. The ratio of CldU to IdU tract lengths is presented, with the median values marked on the graphs and listed at the top. At least 90 tracts were quantified for each sample. Asterisks indicate statistical significance (Mann–Whitney, two-tailed). Schematic representations of the assay conditions are shown at the top. Western blots confirming the ZRANB3 and SMARCAL1 knockdown are shown in Supplementary Fig. S1c,d. h, i Cellular viability assays showing that loss of EXO1 partially suppresses the HU (h) and cisplatin (i) sensitivity of BRCA1- or BRCA2-knockdown HeLa cells, at two different concentrations as indicated. The average of three independent experiments, with standard deviations indicated as error bars, is shown. Asterisks indicate statistical significance (t test, two-tailed, unpaired). Western blots showing EXO1 deletion are shown in Supplementary Fig. S3b. Source data are provided as a Source data file.

Fig 5: Exonucleolytic and endonucleolytic processing of HU and BPA-induced ssDNA gaps by MRE11 and EXO1 leads to DSB formation.a Neutral comet assay showing that DSBs induced by treatment of HeLa-BRCA2KO cells with 0.4 mM HU for 2 h are suppressed by PRIMPOL depletion. At least 70 comets were quantified for each sample. The median values are marked on the graph, and asterisks indicate statistical significance (Mann–Whitney, two-tailed). b, c Neutral comet assays showing that treatment with 0.4 mM HU or 200 µM BPA for 2 h causes DSBs in HeLa-BRCA2KO cells, which are suppressed by inhibition of MRE11 exonuclease activity by mirin or if its endonuclease activity by PFM01 (b), EXO1 knockout (b) or EXO1 knockdown (c). At least 40 comets were quantified for each sample. The median values are marked on the graph, and asterisks indicate statistical significance (Mann–Whitney, two-tailed). d–f γH2AX immunofluorescence showing that that treatment with 0.4 mM HU (d, e) or with 200 µM BPA (f) for 2 h increases γH2AX foci in in HeLa-BRCA2KO cells, which is suppressed by EXO1 knockout (d, f) or by inhibition of MRE11 exonuclease activity by mirin or if its endonuclease activity by PFM01 (e, f). At least 45 cells were quantified for each condition. The mean value is represented on the graphs, and asterisks indicate statistical significance (t test two-tailed, unpaired). g Cellular viability assays showing that loss of EXO1 partially suppresses the BPA sensitivity of BRCA1- or BRCA2-knockdown HeLa cells, at two different concentrations as indicated. The average of three independent experiments, with standard deviations indicated as error bars, is shown. Asterisks indicate statistical significance (t test, two-tailed, unpaired). Source data are provided as a Source data file.