Fig 2: Analysis of BRCA1 antibodies by immunofluorescence in MEF cells. Laser stripes of BRCA1 and ?H2AX at 1h time point after laser micro-irradiation in MEF cells.

Fig 3: BRCA1 phosphorylation controls TOP2B ubiquitination and DNA-binding affinity. (a) Purified TOP2Bubi (ubi) and TOP2Bdeubi (Deu) shown by silver staining and immunoblotting. (b) EMSA comparing TOP2Bubi versus TOP2Bdeubi for their binding affinity to EGR1 TSS no. 3 (–132 to +62). Silver-stained. SM, DNA size marker. (c) Ubiquitinated TOP2B binds to DNA with much higher affinity than deubiquitinated one. A plot summarizing EMSA to derive KD values. (d) Immobilized template assay results. Tight TOP2B association with EGR1 TSS (Pellet, –423 to +332) before NTP addition, regardless of recombinant BRCA1 species added at T1 during PIC formation. (e) Immobilized template assay results. Comparison of the degrees of TOP2B associated (Pellet, P) with and dissociated from EGR1 TSS (Supernatant, S) when WT, SA and SD BRCA1 were added after NTP addition (T2). (f) Quantification of TOP2B in pellet (DNA bound, i) and in supernatant (released, ii) in immobilized template assays. Error bars in s.d. (n = 3). ***p < 0.005, **p < 0.01, *p < 0.05.

Fig 4: Validation of BRCA1 antibodies in Western blot with mouse testis and MEC cells. (A) A representative 8-week-old BRCA1?11/?11 mouse on the right was smaller compared with its WT littermate on the left in the p53+/- background. (B) The spleen and testis isolated from BRCA1?11/?11 and WT littermate male mice at 2-month old. (C) BRCA1 WB with testis tissue lysate from BRCA1?11/?11 and WT littermate male mice at 2-month old. (D) BRCA1 WB with primary mouse mammary epithelial cell lysate from BRCA1?11/?11 and WT littermate female mice at 6-week old.

Fig 5: Reduced BRCA1 and increased 53BP1 occupancy at stalled replication forks.a Representative images (left panel) and quantification of PLA signals (BRCA1: EdU) from U2OS cells transfected with the indicated siRNA after exposure to 4 mM HU for 5 h. The scale bar represents 10 µm. Quantification of PLA signals (BRCA1: EdU) is based on EdU-positive cells. Representative of n = 3 independent experiments. Data are represented as median ± 95% CI (n = 200 cells). ****p < 0.0001 by two-tailed Mann–Whitney test. b ChIP-qPCR analysis for detection of BRCA1 enrichment at CFSs (FRA3B, FRA16D, FRA7.1, and FRA7.2) and control sites (NFS-1, NFS-2) in mock-treated (DMSO) or aphidicolin-treated (APH) U2OS cells transfected with shLSH or shCtrl. Data are represented as mean ± SD (n = 3 independent experiments). ns = not significant, ****p < 0.0001 by Student’s two-tailed t test. c Representative images of PLA signals (53BP1: EdU) from U2OS cells transfected with the indicated siRNA after exposure to 4 mM HU for 5 h. The scale bar represents 10 µm. d Quantification of PLA signals (53BP1: EdU) from U2OS cells as treated in Fig. 6C. Quantification of PLA signals (53BP1: EdU) is based on EdU-positive cells. Representative of n = 3 independent experiments. Data are represented as median ± 95% CI (n = 200 cells). ****p < 0.0001 by two-tailed Mann–Whitney test. e ChIP-qPCR analysis for detection of 53BP1 enrichment at CFSs (FRA3B, FRA16D, FRA7.1, and FRA7.2) and control sites (NFS-1, NFS-2) in mock-treated (DMSO) or aphidicolin-treated (APH) U2OS cells transfected with shLSH or shCtrl. Data are represented as mean ± SD (n = 3 independent experiments). *p < 0.05, **p < 0.01 by Student’s two-tailed t test. f Nascent DNA degradation analysis after exposure to HU in U2OS cells transfected with siCtrl or siLSH or a combination of siLSH and si53BP1 leading to an intermediate 53BP1 depletion (0.3 in Supplementary Fig. 13B). The IdU/CldU ratio calculations are done as outlined in Fig. 2a. Representative of n = 3 independent experiments. Data are represented as median ± 95% CI (n = 250 DNA fibers). ****p value < 0.0001 by two-tailed Mann–Whitney test. g ChIP-qPCR analysis for detection of BRCA1 enrichment at indicated CFSs in aphidicolin treated (APH) U2OS cells transfected with shLSH and either siCtrl or si53BP1. Data are represented as mean ± SD (n = 3 independent experiments). ****p < 0.01 by Student’s two-tailed t test.