Fig. 4: Targeting of RAD51 to ssDNA by CTRB and mini-BRCA2. | Nature Communications

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

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

Fig. 4

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.

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