Fig. 2: BRCA2 NTD and C-terminal domains contribute to BRCA2 location at nascent DNA.

a (Top left) Scheme of the assay. (Top right): Representative images of in situ PLA on nascent DNA between biotinylated EdU detected with anti-biotin antibody and BRCA2-specific antibody in DLD1 BRCA2-deficient cells (BRCA2^−/−) stably expressing either BRCA2 WT or the variants C315S (A7), S273L (C5), R3052W, and S3291A mutant, as indicated. Cells left untreated (UNT) or treated with HU (30′ or 1 h 0.2 mM) are shown. An individual signal is observed (focus) if the two probed proteins (BRCA2 and EdU-Biotin) are in close proximity (<40 nm). For all the experiments we carried out two single-antibody control (anti-BRCA2 and anti-biotin) to assess the specificity of the PLA signal. The scale bar indicates 10 μm. (Bottom) Quantification of the BRCA2 recruitment was measured as the number of PLA foci observed per nucleus. The data represent the mean + SEM with 200–300 cells analyzed in each experimental data set at each time point. The number of independent experiments performed was as follows: BRCA2 WT: (UNT n = 10; 5′ n = 5; 30′ n = 8; 1 h n = 7), C315S: (UNT n = 6; 5′ n = 3; 30′ n = 5; 1 h n = 5); S273L:(UNT n = 4; 30′ n = 2; 1 h n = 2); S3291A: (UNT n = 3; 5′ n = 2; 30′ n = 2; 1 h n = 2); R3052W: (UNT n = 6; 5′ n = 3; 30′ n = 3; 1 h n = 3). Statistical difference was determined by the Kruskal–Wallis test followed by Dunn’s multiple comparison test. The p-values show significant differences compared to the BRCA2 WT clone. ns, not significant (p = 0.7539 in UNT BRCA2 WT vs. R3052W), *p = <0.05 (p = 0.0136 at 5′ BRCA2WT vs. R3052W), **p < 0.01 (p = 0,0042 at 1 h BRCA2WT vs. R3052W), ***p < 0.001 (p = 0.0002 at 30′ BRCA2WT vs. R3052W), ****p < 0.0001). b (Left) Scheme of the assay and representative images of in situ PLA on nascent DNA between biotinylated EdU detected with anti-biotin antibody and BRCA2-specific antibody in DLD1 BRCA2 WT cells after 4 h Thymidine chase in cells left untreated or treated with HU (30′ 0.2 mM). (Right) Quantification of the BRCA2 recruitment measured as the number of PLA foci observed per nucleus after 4 h Thymidine chase in BRCA2 WT cells at different time points. The data represent the mean + SEM of two independent experiments with 200–300 cells analyzed in each experimental data set at each time point. Schemes of the PLA assay created with BioRender.com. Source data are provided as a Source Data file.