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

Abstract

The tumor suppressor BRCA2 participates in DNA double-strand break repair by RAD51-dependent homologous recombination and protects stressed DNA replication forks from nucleolytic attack. We demonstrate that the C-terminal Recombinase Binding (CTRB) region of BRCA2, encoded by gene exon 27, harbors a DNA binding activity. CTRB alone stimulates the DNA strand exchange activity of RAD51 and permits the utilization of RPA-coated ssDNA by RAD51 for strand exchange. Moreover, CTRB functionally synergizes with the Oligonucleotide Binding fold containing DNA binding domain and BRC4 repeat of BRCA2 in RPA-RAD51 exchange on ssDNA. Importantly, we show that the DNA binding and RAD51 interaction attributes of the CTRB are crucial for homologous recombination and protection of replication forks against MRE11-mediated attrition. Our findings shed light on the role of the CTRB region in genome repair, reveal remarkable functional plasticity of BRCA2, and help explain why deletion of Brca2 exon 27 impacts upon embryonic lethality.

Fig. 1. Bioinformatic and NMR analyses of BRCA2 and the CTRB.

a (TOP) Schematic highlighting the location of different functional regions in BRCA2. (BOTTOM) The degree of conservation throughout full-length BRCA2 in selected vertebrates. Of note, the alignment score does not match the number of residues in human BRCA2 as a result of a substantial amount of indels in the BRCA2 sequence. For orientation, we have annotated the sequence position for human BRCA2 in the schematic on top of the conservation graph. Source data are provided as a Source Data file. b Evolutionary divergence for five different example regions of BRCA2: disordered: residues 296–387, BRC4 repeat: residues 1517–1548, OB1: residues 2718–2800, Tower domain: residues 2846–2950, CTRB core fragment: 3260–3337. Source data are provided as a Source Data file. c ¹H-¹⁵N-HSQC HSQC of the CTRB polypeptide in the absence (black) or presence (red) of a 12-mer ssDNA. The ¹H-¹⁵N-HSQC NMR spectrum was recorded using 20 μM uniformly ¹H-¹⁵N-labeled BRCA2 CTRB (3260–3337) in 1 x PBS, pH 6.0, 10 mM DTT, at 5 °C. The full ¹H-¹⁵N- HSQC spectrum can be found in Supplementary Fig. 1c. d Relative change in peak intensities of the HSQC cross-peaks in the presence and absence of ssDNA. Two regions, designated A and B, where the effect of ssDNA is most prominent, are highlighted. The noise of the signals was ±5.5%. Source data are provided as a Source Data file.

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.

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

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.

Fig. 4. Targeting of RAD51 to ssDNA by CTRB and mini-BRCA2.

a (i) Schematic of the assay. The magnetic resin containing ssDNA was incubated with RAD51 and CTRB (ii), or mini-BRCA2/DSS1 (iii) with and without an excess of radiolabeled dsDNA added as a protein trap. Proteins bound to the ssDNA (Beads) were eluted and analyzed alongside the reaction supernatant(Supernatant) by SDS-PAGE and Coomassie blue staining. The results were quantified and graphed. Phosphorimaging analysis of the dried gel verified that the radiolabeled dsDNA trap remained in the Supernatant fraction. (*, no dsDNA trap; **, GST-DSS1) (n = 3 biologically independent experiments; mean value ± SD; one-way ANOVA and Dunnett’s multiple comparison test; ***p < 0.001; ****p < 0.0001. p value between the CTRB WT group and the control (ctr) group is 1.172 × 10⁻⁴; for the CTRB-F/A group is 1.512 × 10⁻⁴; for the CTRB 4 A group is 4.127 × 10⁻⁴; between the mini-BRCA2 WT group and the 4 A group is 0.01079.) Source data are provided as a Source Data file. b Model for CTRB function in RAD51 presynaptic filament assembly. Through its interaction with oligomeric RAD51 and DNA engagement, the CTRB makes a crucial contribution toward the timely assembly of the RAD51 presynaptic filament.

Fig. 5. Significance of CTRB attributes in DNA damage repair.

a Clonogenic survival of DLD1 cells expressing wild-type BRCA2 or BRCA2 variants with the indicated CTRB mutation (F/A, 4 A, or F/A-4A) after treatment of MMC or Rucaparib. EV: empty vector. (n = 4 biologically independent experiments for MMC, mean value ± SEM; n = 3 biologically independent experiments for Rucaparib, mean value ± SD). Source data are provided in the Source Data file. b HR proficiency was measured using the DR-GFP gene conversion reporter in HeLa cells depleted for endogenous BRCA2 and expressing BRCA2 or BRCA2 variants with the indicated CTRB mutation. Ctr (control): not treated. (n = 5 biologically independent experiments; mean value ± SD; unpaired t-test (two-tailed); *p = 4.928 × 10⁻²; **p < 0.01; ***p < 0.001; ****p < 0.0001. p value between the WT group and the EV group is 1.608 × 10⁻⁴; for the F/A group is 1.116 × 10⁻³; between the F/A group and the F/A_4 A group is 2.898 × 10⁻³; between the 4 A group and the FA_4 A group is 7.481 × 10⁻⁴). Source data are provided as a Source Data file. c HR proficiency was measured in DLD1 cells expressing BRCA2 and BRCA2 variants with the indicated CTRB mutation using a CRISPR/Cas9-based gene targeting assay. Ctr (control): not treated. (n = 3 biologically independent experiments; mean value ± SEM; one-way ANOVA and Tukey’s multiple comparison test; **p = 1.625 × 10⁻³; ****p < 0.0001. p value between the WT group and the 4 A group is 1.183 × 10⁻⁵.) Source data are provided as a Source Data file. d RAD51 focus formation after MMC treatment. Representative micrographs of RAD51 foci from Supplementary Fig. 4c are shown. (n = 7 biologically independent experiments for WT, n = 4 for EV and 4 A, n = 3 for F/A and F/A_4 A; mean value ± SEM; one-way ANOVA and Tukey’s multiple comparison test; ns (no significant difference): p = 0.2212; ****p < 0.0001. p value between the WT group and the F/A group is 7.100 × 10⁻⁵; between the 4A group and the F/A_4A group is 8.623 × 10⁻⁵.) Source data are provided as a Source Data file.

Fig. 6. Significance of CTRB attributes in replication fork protection.

a Schematic of DNA fiber analysis (i) and representative micrographs (ii). Dot plots of IdU to CldU tract length ratios for individual replication forks in unperturbed, and HU-treated cells with or without mirin (iii, iv). The median value of 100–150 CldU and IdU tracts from three and four independent experiments for unperturbed and treated cells, respectively, is indicated. Kruskal–Wallis test followed by Dunn’s multiple comparisons test. (ns (no significant difference): p ≥ 0.05; *p < 0.05; ****p < 0.0001. p value between the EV group and the EV: HU + Mirin group is 0.3046; between the F/A group and the F/A: HU + Mirin group is 0.09772; between the 4A group and the 4A:HU + Mirin group is 4.396 × 10⁻²; between the F/A_4A group and F/A_4A:HU + Mirin group is 0.04908.) Representative micrographs of DNA fibers (EV: empty vector. WT BRCA2 cells) from Supplementary Fig. 5 are shown. Source data are provided as a Source Data file. b Model for CTRB function at regressed replication forks. Through its interaction with oligomeric RAD51 and DNA engagement, the CTRB helps seed the assembly of a RAD51 protein filament of the arm of a regressed replication fork that harbors the free DNA end.