Fig. 4: RAD51C Walker B variant impact on DNA binding, ATPase activity, and CX3 complex formation. | Nature Communications

Fig. 4: RAD51C Walker B variant impact on DNA binding, ATPase activity, and CX3 complex formation.

From: Comprehensive RAD51C ovarian cancer variant analysis uncouples homologous recombination and replicative functions

Fig. 4

a RAD51C-L238R cannot be stably purified in the BCDX2 complex. Ni-NTA and α-FLAG affinity purification of BCDX2 complexes harboring either wild-type or L238R variant of RAD51C. The lysate, flow through (FT), and elution was run on an SDS-PAGE gel and RAD51B-RAD51C (B/C) and RAD51D-XRCC2 (D/X2) subcomplexes are indicated. b RAD51C-V239G and A279P can be purified in the BCDX2 complex. Flow chart summarizing BCDX2 purification and Coomassie blue stained SDS-PAGE gel showing fractions of size exclusion chromatography for purification of BCDX2 complexes containing wild-type RAD51C or the indicated RAD51C Walker B variants. RAD51B-RAD51C (B/C) and RAD51D-XRCC2 (D/X2) subcomplexes are indicated. c BCA279PDX2 shows reduced DNA binding activity. ssDNA binding by the BCDX2 complexes containing wild-type RAD51C or the indicated RAD51C Walker B variants at increasing DNA concentrations (nM) assessed by electrophoretic mobility shift assays using an 80-nucleotide substrate. d BCV239GDX2 shows reduced ATPase activity. Quantification of ssDNA binding by the BCDX2 complexes from three independent experiments plotted as mean values ± s.d. e ATP hydrolysis with 1 μM BCDX2 complexes containing WT and indicated RAD51C Walker B variants with or without ssDNA (phix174) were assessed at the indicated time points (min). f Quantification of ATP hydrolysis measurements for three independent experiments plotted as mean values ± s.d. g RAD51C-V239G can be purified in the CX3 complex, but RAD51C-L238R and A279P cannot. CX3 complex formed with MBP tagged WT or the indicated RAD51C Walker B mutants and FLAG tagged XRCC3 were affinity purified from amylose resin followed by FLAG resin and loaded onto a Superdex200 increase 10/300 size exclusion column. Fractions were run on an SDS-PAGE gel and visualized by Coomassie staining, and void and monodispersed fractions are indicated. Gel images in Fig. 4a–c, g are representative of at least two independent experiments.

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