Fig. 2: HR mediator activity of the CTRB and relevance of RAD51 interaction and DNA binding.

a The CTRB region in BRCA2. Alignment of the domain in BRCA2 orthologs and the locations of the ³²⁹⁸F to A and ³²⁶⁶KKRR to AAAA mutations. b SDS-PAGE of purified wild-type CTRB, CTRB-F/A mutant, and CTRB-4A mutant. The experiment was repeated three times with similar results. Source data are provided as a Source Data file. c Binding of ssDNA and dsDNA by CTRB, CTRB-F/A, or CTRB-4A as examined by EMSA. Quantification of the DNA binding results is shown in Supplementary Fig. 2a. 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 S-tag pulldown for analysis of RAD51-CTRB interaction; RAD51 and CTRB (4 µg each) were tested. S: Supernatant containing unbound proteins, W: Wash, E: SDS-eluate. Note that the CTRB-F/A mutant is defective in RAD51 interaction, while the CTRB-4A mutant is proficient in this regard. The samples were derived from the same experiment and the gels were processed in parallel. The experiment was repeated three times with similar results. Source data are provided as the Source Data file. e DNA strand exchange assay with RAD51 and CTRB. (i) Reaction schematic and (ii) CTRB (lanes 3 to 6: 0.6, 1.2, 2.3, and 3 μM) was tested with ssDNA pre-incubated with RAD51 (2 μM). Lane 7 contained 3 μM CTRB and the results were quantified and graphed in (iii). (n = 3 biologically independent experiments; mean ± SD; one-way ANOVA and Tukey’s multiple comparison test; ****p =  9.280 × 10⁻⁶). Source data are provided as a Source Data file. f Testing of HR mediator activity of the CTRB. (i) Reaction schematic and (ii) CTRB (lanes 4 to 8: 0.4, 0.8,1.6, 2.4, 3 μM) was tested with RAD51 (2 µM) in the DNA strand exchange reaction with ssDNA precoated with RPA (600 nM) and the results were quantified and graphed in (iii). (n = 3 biologically independent experiments; mean ± SD; one-way ANOVA test and Tukey’s multiple comparison test; ns (no significant difference): p = 0.8620; ****p = 7.180 × 10⁻⁵)). Source data are provided as a Source Data file. g The CTRB-F/A and CTRB-4A mutants (3 μM each) were examined as indicated. (n = 5 biologically independent experiments for WT and F/A, n = 3 for 4 A; mean ± SD; one-way ANOVA and Tukey’s multiple comparison test; ns (no significant difference): p ≥ 0.05; **p = 0.0069; ****p < 0.0001; p value between the RAD51 only group and the RAD51 + RPA + CTRB WT group is 0.9999; for the RAD51 + CTRB-F/A group is 0.9992; for the RAD51 + CTRB 4 A group is 0.9737). The samples were derived from the same experiment and the gels were processed in parallel. Source data are provided as a Source Data file.