Fig. 3: Contribution of CTRB functional attributes to HR mediator activity of BRCA2. | Nature Communications

Fig. 3: Contribution of CTRB functional attributes to HR mediator activity of BRCA2.

From: DNA binding and RAD51 engagement by the BRCA2 C-terminus orchestrate DNA repair and replication fork preservation

Fig. 3

a Schematic of BRCA2 species tested. b SDS-PAGE of purified BRC4-DBD, DBD-CTRB, and BRC4-DBD-CTRB complexed with GST-DSS1. The experiment was repeated three times with similar results. Source data are provided as a Source Data file. c HR mediator activity of BRCA2 species. (i) Schematic of the DNA strand exchange assay. The indicated BRCA2 species (60, 120, 180, 240, and 300 nM) were tested for HR mediator activity (ii) and the results were quantified and graphed in (iii). (n = 4 biologically independent experiments; mean value ± SD; one-way ANOVA and Tukey’s multiple comparison test; ns (no significant difference): p ≥ 0.05; ****p < 0.0001. p value between the RAD51 only group and the RAD51 + RPA + mini-BRCA2 (300 nM) group is 0.7872; between DBD-CTRB and BRC4-DBD is 0.9999.) The samples were derived from the same experiment and the gels were processed in parallel. Source data are provided as a Source Data file. d Schematic of mini-BRCA2 species that harbor the CTRB-F/A or CTRB-4A mutation. e Anti-Flag affinity pulldown assay to test for the interaction of mini-BRCA2/DSS1 (WT, CTRB-F/A, or CTRB-4A, 3 μg each) and RAD51 (8 μg). S Supernatant containing unbound proteins, W Wash, E SDS-eluate. The experiment was repeated three times with similar results. Source data are provided as a Source Data file. f Binding of ssDNA and dsDNA by mini-BRCA2/DSS1. (i) WT and CTRB-4A, (ii) WT and CTRB-F/A. Note that the same wild-type DNA binding data were plotted in panels (i) and (ii) for comparison with the 4 A mutant (i) or F/A mutant (ii). (n = 3 biologically independent experiments; mean value ± SD). The representative gel images are shown in Supplementary Fig. 3b. Source data are provided as a Source Data file. g (i) Schematic of the DNA strand exchange assay. (ii) HR mediator activity of mini-BRCA2/DSS1 (WT, CTRB-F/A, CTRB-4A, or CTRB-F/A-4A at 100 and 300 nM) was graphed. (n = 5 (sample number from four biologically independent experiments) for RAD51, RPA + RAD51; n = 3 for 100 nM WT, 300 nM 4 A; n = 4 for the rest of these groups); mean value ± SD; one-way ANOVA and Tukey’s multiple comparison test; ns (no significant difference): p = 0.9999; *p = 0.01219; **p = 4.365 × 10−3; ****p < 0.0001). Representative gel images are shown in Supplementary Fig. 3d. Source data are provided as a Source Data file.

Back to article page