Fig 2: WASp, N-WASp and DIAPH1 associate with replication forks. (A) STRING analysis of iPOND/MS data in untreated and HU treated HeLa S3 cells. (B) Representative images and dot plots of the number of N-WASp/Biotin (nascent DNA) and WASp/Biotin (Nascent DNA) PLA foci per nucleus in HeLa cells 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 negative Ctrl N-WASp ab only [0] n = 524, NT [1] n = 731, 1 mM/1 h [2] n = 594, 4 mM/3 h [2] n = 561; right panel median values Negative Ctrl WASp ab only [0] n = 545, NT [0] n = 649, 1 mM/1 h [1] n = 557, 4 mM/3 h [0] n = 620). (C) Representative images and dot plots of number of N-WASp/Biotin (Nascent DNA) and WASp/Biotin (Nascent DNA) PLA foci per nucleus in HeLa cells treated with 1 mM HU for 1 h, followed or not by thymidine chase as indicated (red lines indicate mean values). Dot plots represent data pooled from two independent experiments. Statistical significance was determined using the Mann–Whitney test (Left panel median values 1 mM/1 h [4] n = 139, 1 mM/1 h Thy [2] n = 244; right panel median values 1 mM/1 h [5] n = 1012, 1 mM/1 h Thy [0.5] n = 1084).

Fig 3: Depletion of WASp, N-WASp or DIAPH1 leads to defects in ATR signalling activation. (A) Dot plots of number of RAD51/Biotin (nascent DNA) PLA foci per nucleus in HeLa cells treated with control siRNA or siRNA against WASp upon treatment with 4 mM HU for 3 h. Dot plots represent data pooled from two independent experiments (red lines indicate mean values). Statistical significance was determined using the Mann–Whitney test (left panel median values siCTRL NT [7] n = 145, siCTRL 4 mM/3 h [12] n = 147, siWASp NT [3] n = 151, siWASp 4 mM/3 h [3] n = 129; right panel median values siCTRL NT [4] n = 393, siCTRL 4 mM/3 h [9.5] n = 516, siDIAPH1 NT [4] n = 545, siDIAPH1 4 mM/3 h [7] n = 518. (B). WB showing CHK1 phosphorylation in HeLa cells with or without indicated HU treatment for 1 h, treated with control siRNA (siCTRL) or siRNA targeting N-WASp or WASp as well as the same analysis in DIAPH1-deficient patient-derived cells and the corresponding control cells. Bar charts represent mean ± SEM of three experiments and statistical significance was determined using t test. (C) Quantification of the frequency of origin firing in HeLa cells treated with control siRNA or siRNA against N-WASp, WASp or DIAPH1. Bar charts represent mean ± SEM of four (siN-WASp and siWASp) or three (DIAPH1) independent experiments. Statistical significance was determined using t test.

Fig 4: 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 5: WASp, N-WASp, ARP2/3 and DIAPH1 are required for intact replication dynamics under condition of replicative stress. (A) Representative DNA fibre images and box plots of CldU/IdU tract ratios from HeLa cells treated with control siRNA (–) or siRNA against WASp, N-WASp or DIAPH1(+) and exposed to 1 mM HU during CldU labelling pulse. Whiskers indicate 5–95 percentile. Box plots represent data pooled from three (siWASp) or two (siN-WASp and siDIAPH1) independent experiments. Statistical significance was determined using the Mann–Whitney test (left panel siCTRL n = 223; siN-WASp n = 218; central panel siCTRL n = 222; siDIAPH1 n = 218; right panel siCTRL n = 311, siWASp n = 282). (B) Box plots of CldU/IdU tract ratios from DIAPH1-deficient patient-derived cells treated with 1 mM HU during CldU labelling pulse. Whiskers indicate 5–95 percentile. Box plots represent data pooled from three independent experiments. Statistical significance was determined using the Mann–Whitney test (DIAPH1-/-+DIAPH1 n = 379; DIAPH1-/- n = 374). (C). Box plots of CldU/IdU tract ratios of HeLa cells treated with control siRNA (–) or siRNA against N-WASp, DIAPH1 or WASp (+). After IdU pulse, cells were exposed to 2 mM HU for 2 h followed by release into CldU for 60 min. Whiskers indicate 5–95 percentile. Box plots represent data pooled from two independent experiments. Statistical significance was determined using the Mann–Whitney test (left panel siCTRL n = 166; siN-WASp n = 64; central panel siCTRL n = 199; siDIAPH1 n = 205; right panel siCTRL n = 204, siWASp n = 198). (D) Box plots of CldU/IdU tract ratios from DIAPH1-deficient patient-derived cells treated with CK-666 (ARP2/3 inhibitor) (+) or DMSO (–). During CldU labelling pulse, cells were exposed to 1 mM HU. Whiskers indicate 5–95 percentile. Box plots represent data pooled from two independent experiments. Statistical significance was determined using the Mann–Whitney test (DIAPH1−/−+DIAPH1 n = 210; DIAPH1−/−+DIAPH1 + ARP2/3i n = 173; DIAPH1−/−n = 245, DIAPH1−/−+ARP2/3i n = 211).