Fig 2: Isobavachalcone (IBC) impedes DNA end resection and RAD51 foci formation.(A) MCF-7 cells were treated with DMSO or 15 μM IBC for 1 hr, then camptothecin (CPT, 1 μM) was added for 2 hr. The cells were fixed immediately or washed and allowed to recover in medium containing DMSO or IBC for 24 hr before fixation. 53BP1 foci in p27-positive nuclei were detected by immunofluorescence microscopy. Representative images are shown. Bar: 5 μm. (B) 53BP1 foci number was quantified using CellProfiler. Representative data from three independent experiments are shown. ****p<0.0001; ns, not significant, Mann–Whitney rank sum test. (C) MCF-7 cells were treated with DMSO or 15 μM IBC for 2 hr, then CPT (1 μM) was added for 2 hr, as indicated. Cells were fractionated into cytosol and nuclei (chromatin) and RPA in both fractions was detected by western blotting. Actin and TBP were used as markers of cytosol and chromatin fractions, respectively. The fold change of the chromatin-bound RPA signal relative to the DMSO control was quantified. The p-value was determined using unpaired t-test (n = 3). (D) MCF-7 cells were incubated with 10 µM BrdU for 24 hr to label genomic DNA, then DMSO, 15 µM IBC or the CHK2 inhibitor BML-277 (20 μM) were added for 2 hr followed by addition of 5 μg/ml bleomycin for 1 hr. DNA fibers were spread on glass slides and BrdU was detected by immunofluorescence microscopy without DNA denaturation. The length of BrdU the tracks was measured and the median for each condition is indicated in red. At least 250 fibers were measured for each condition. Median of two independent experiments is indicated in red (n = 2). (E) MCF-7 cells were incubated with 10 µM BrdU for 24 hr to label genomic DNA in the presence of DMSO or IBC then exposed to ionizing radiations (8 Gy). Cells were collected 60 or 120 min after irradiation and BrdU tracks measured as in (B). ****p<0.0001, Mann–Whitney rank sum test. (F) DIvA cells were treated with either DMSO or IBC for 2 hr then DNA breaks were induced by treatment with 300 nM 4-hydroxytamoxifen (4-OHT) for 4 hr. Resection at two break sites, DSB-II and DSB-V, was determined as the percentage of ssDNA at these sites, calculated as indicated in the ‘Materials and methods’. Data are means ± SD (n = 3). The p-values are indicated (two-tailed paired t-test). (G) MCF-7 cells were treated with DMSO, IBC, or BML-277 for 2 hr, then irradiated as described above. After 1 hr, RAD51 foci were detected by CSK-immunofluorescence microscopy and foci number was quantified using CellProfiler. Representative data (left) and immunofluorescence images (right) from two independent experiments are shown. Bar: 10 μm. ****p<0.0001, Mann–Whitney rank sum test. Figure 5—source data 1.Original membranes corresponding to Figure 5C with labels.Cytosol for cytosol fraction. Chr for chromatin fraction. Alpha-actin was used as a marker of cytosolic fraction. TBP was employed as a marker of chromatin fraction. The molecular weight markers are indicated. Figure 5—source data 2.Original membranes corresponding to Figure 5C.

Fig 3: Depletion of WASp, N-WASp or DIAPH1 leads to increased degradation of nascent DNA and defects in RPA recruitment to perturbed replication forks. (A) Box plots of CldU/IdU tract ratios from HeLa cells treated with control siRNA(-) or siRNA targeting N-WASp, DIAPH1 or WASp (+). CldU/IdU pulse labelling was followed by treatment with 4 mM HU for 4h. Whiskers indicate 5–95 percentile. Box plots represent data pooled from four (siWASp), or two (siN-WASp, siDIAPH1) independent experiments. Statistical significance was determined using the Mann–Whitney test (left panel siCTRL n = 123; siN-WASp n = 116; central panel siCTRL n = 210; siDIAPH1 n = 204; right panel siCTRL n = 232, siWASp n = 259). (B). Box plots of CldU/IdU tract ratios from HeLa cells treated with control siRNA or siRNA targeting N-WASp, DIAPH1 or WASp and SMARCAL1. CldU/IdU pulse labelling was followed by treatment with 4 mM HU for 4 h. Whiskers indicate 5–95 percentile. Box plots represent data pooled from two independent experiments. Statistical significance was determined using the Mann–Whitney test (siCTRL n = 452, siSMARCAL1 n = 499, siDIAPH1 n = 235, siDIAPH1/siSMARCAl1 n = 248, siN-WASp n = 231, siN-WASp/siSMARCAL1 n = 235, siWASp n = 232, siWASp/siSMARCAL1 n = 230). (C) Representative images and dot plots of RPA signal intensity per nucleus of Hela cells treated with control siRNA or siRNA targeting N-WASp, WASp or DIAPH1 upon HU treatment as indicated (red lines indicate mean values). Dot plots represent data pooled from three independent experiments. Statistical significance was determined using the Mann–Whitney test (left panel median values siCTRL NT [0.4295] n = 300, siCTRL 1 mM/1 h [2.4368] n = 300, siCTRL 4 mM/3 h [6.1924] n = 300, siN-WASp NT [0.1678] n = 300, siN-WASp 1 mM/1 h [0.2192] n = 300, siN-WASp 4 mM/3 h [0.4568] n = 300; middle panel median values siCTRL NT [1.2417] n = 373, siCTRL 1 mM/1 h [1.3593] n = 395, siCTRL 4 mM/3 h [3.9828] n = 407, siDIAPH1 NT [1.1798] n = 392, siDIAPH1 1 mM/1 h [1.1713], siDIAPH1 4 mM/3 h [2.6214] n = 439; right panel median values siCTRL NT [0.5790] n = 300, siCTRL 1 mM/1 h [0.8026] n = 300, siCTRL 4 mM/3 h [1.7282] n = 300, siWASp NT [0.5426] n = 300, siWASp 1 mM/1 h [0.4892] n = 300, siWASp 4 mM/3 h [0.5701] n = 300.

Fig 4: The accumulation of DNA damage is sensitive to abundance changes of MCM2-7 subunits.(a) Levels of total and chromatin-bound MCM2 and MCM7 in parental HCT116 upon siRNA-mediated depletion of MCM2. (b) Accumulation of 53BP1 and (c) anaphase bridges in HCT116 upon depletion of MCM2 with and without replication stress. All plots show mean±s.e.m. of three independent experiments, at least 500 cyclin A-negative cells or 50 anaphases were scored in each experiment. (d) Survival rates of HCT116, RPE1 and their trisomic and tetrasomic derivatives upon overexpression of wild-type and mutant alleles of MCM2 (MCM-457A). (e) Survival rates of HCT116, RPE1 and the trisomic and tetrasomic derivatives upon overexpression of wild-type and mutant alleles of ORC1 (ORC1ΔBAH). (f) Survival rates of HCT116, RPE1 and the trisomic and tetrasomic derivatives upon overexpression of wild-type and mutant alleles of RPA1 (RPA1 L221P). Survival rates were normalized to the control (pcDNA transfected sample). All plots show mean+s.e.m. of three independent experiments; nonparametric t-test; *P<0.05, **P<0.01, ***P<0.001. (g) Levels of total and chromatin-bound MCM2 and MCM7 in HCT116 5/3 upon transient overexpression of MCM7. (h) Formation of 53BP1 foci and (i) accumulation of anaphase bridges in HCT116 and HCT116 5/4 upon transient overexpression of MCM7. One representative plot of three independent experiments is shown. Nonparametric t-test; *P<0.05, **P<0.01, ***P<0.001.

Fig 5: DNA2 and RPA1 promote resection resulting in a-HDR or mutagenesis at nicks.(A) Alignment of RPA1 protein sequences in the conserved N-terminal region to which the S. cerevisiae Rfa1-t11 (K45E) mutation maps [22]. (B) Frequencies of a-HDR at nicks or c-HDR at DSBs as determined by reporter assays in 293 TL cells ectopically expressing RPA1 WT or its mutant derivatives. Frequency values represent the mean ± SEM from a representative experiment; *** indicates p<0.001 for the frequency difference between the indicated sample and sample expressing RPA1-WT. (C) Frequencies of a-HDR at nicks targeted to the CD44 gene in U2OS cells ectopically expressing RPA1-WT or its mutant derivatives and provided with a ssDNA donor for a-HDR by sequence analysis. (D) Maps of the fractional decrease in the number of base calls at each position within the indicated region spanning the gRNA 4 cleavage site in U2OS cells in cells treated as indicated and lacking or provided with a ssDNA donor complementary to the nicked strand (cN) to support a-HDR. (E) Maps of positions and spectra of SNVs within the indicated 65 bp region spanning nicks targeted by gRNA 4 to the CD44 gene in U2OS cells treated as indicated. The colored bar represents SNVs at each reference position: A, gray; C, orange; G, blue; T, red.