Fig 2: Knockdown of ?40p53 increases DNA damage following DOX treatment at early time points.A ?H2AX + ve cells after treatment with DOX (3 and 6 h) and normalised by the 0 h in the MCF-7 (-shNT, -sh?40p53, -shp53a, LeGO and ?40p53) and ZR75-1 (-shNT, -sh?40p53, -shp53a) sublines. ?H2AX (1:50) primary antibody was used. Data shown represent three independent experiments of three technical replicates. B BRCA1 and C RAD51 expression after treatment with vehicle (water) or DOX (3 h) in the MCF-7 (-shNT, -sh?40p53, -shp53a, LeGO and ?40p53) and ZR75-1 (-shNT, -sh?40p53, -shp53a) sublines measured by immunofluorescence. BRCA1 (1:100) and RAD51 (1:100) primary antibodies were used. Data shown represent three independent experiments of three technical replicates. Results are shown as the mean ± SD. Statistical analysis were carried out using two-way ANOVA followed by Sidak’s post-test. Results were considered significant at p < 0.05; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 3: Gene expression of p53 interactors in the MCF-7 sublines.A–D RNA-seq validation. Gene expression after DOX treatment (24 h) in the MCF-7 (-shNT, -sh?40p53, -shp53a, LeGO and ?40p53) sublines. A MELK, B NDC80, C UHRF1 and D TRIP13. Data shown represent three independent experiments of three technical replicates. Results are shown as the mean ± SD and were normalised by the relative expression of the vehicle-treated cells. Statistical analyses were carried out using two-way ANOVA followed by Sidak’s post-test. Results were considered significant at p < 0.05; *p < 0.05, **p < 0.01. E Interaction between differentially expressed genes (DEGs) and p53 pathway proteins based on STRING data of high confidence (overall interaction score >0.9; see Supplementary Table S9 for breakdown of score). Orange – p53 pathway proteins; Blue: DEGs in DOX-treated MCF-7-sh?40p53 vs MCF-7-shNT; Green – DEGs in DOX-treated MCF-7-?40p53 vs MCF-7-LeGO; Yellow – DEGs in DOX-treated MCF-7-sh?40p53 vs MCF-7-shNT and MCF-7-?40p53 vs MCF-7-LeGO. F Heatmaps of mRNA levels of p53 interactors: ATM, ATR, CHECK2, HDM2, MAPK1, EP300, TOPOIIa, TOPOIIß, BRCA1, RAD51, RAD52, LIG4, PARP1, APEX1 and OGG1 after 6 or 24 h of DOX treatment and normalised to vehicle-treated cells in the MCF-7 (-shNT, -sh?40p53, -shp53a, LeGO and ?40p53) sublines (see Supplementary Fig. 7, 8 for comparison between sublines and statistical analyses). Data shown represent three independent experiments of three technical replicates.

Fig 4: I-PpoI-induced DSBs selectively recruit HR repair factors to nucleolar caps. (A) BRCA1 and 53BP1 were individually observed at ?H2AX-positive nucleolar caps in I-PpoI transfected cells. (B) Antibody staining of I-PpoI transfected cells shows that HR factors RPA2 (total and phospho-S4/S8), Rad51, and Rad52 are highly enriched at nucleolar caps. (C) 3D immunoFISH was performed on I-PpoI transfected RPE1 cells using a DJ probe, Treacle, and RPA2 antibodies. The insets show enlarged individual caps containing a single NOR.

Fig 5: SFXN4 KD decreases Fe-S containing DNA repair proteins, RAD51, and DNA repair capacity. (A) Western blot of Fe-S containing DNA repair proteins in control and SFXN4 knock-down ovarian cancer cells (siRNA was used to knock down SFXN4 in COV362 cells and CRISPER/Cas was used to knockout SFXN4 KO in OVCAR3 cells). Uncropped images are presented in Supplemental data (Supplemental Figs. S11, S12). (B) Representative immunofluorescence image of RAD51 foci (red) in siRNA SFXN4 knock-down and siRNA control COV362 cells (nuclei were stained with DAPI) and quantification of fold change in RAD51 foci in SFXN4 knockdown versus control cells treated without or with 20 µM cisplatin for 48 h (means and standard deviations, **p < 6.4E-05). Scalebar is 10 µm. (C) DNA repair capacity as measured by dual luciferase assay in siRNA control and siSFXN4 COV362 cells transfected with undamaged or cisplatin damaged luciferase plasmid for 24 h. Shown are means and standard deviations of 3 independent experiments, each with 4 replicas (*p < 4E-04).