Fig. 4: X3C targets RAD51 to ssDNA by restraining dsDNA binding.
From: Structural insight into how RAD51 paralog exchange regulates RAD51 filament formation
a,b, Gel images (a) and quantifications (b) of 80-nt ssDNA (top), 80-bp dsDNA (middle) and 60-bp RNA–DNA hybrid (bottom) binding of RAD51 and RAD51–X3C at the indicated RAD51 concentrations in the presence of AMP-PNP, assessed by EMSAs. Results from four independent experiments are plotted as the mean values ± s.d. c, Top: scheme of ssDNA EMSA for protein preincubated with an eightfold excess of phiX174 circular dsDNA (1.24 μM bp). The asterisk indicates Cy5 label on 80 nt ssDNA. Middle: ssDNA binding of RAD51 and RAD51–X3C at the indicated RAD51 concentrations in the presence of 1 mM AMP-PNP following preincubation with excess circular dsDNA. Bottom: results from three independent experiments are plotted as the mean values ± s.d. d, Top: reaction schematic. Middle: strand exchange with increasing concentrations of either RAD51 alone or RAD51–X3C in presence of AMP-PNP after preincubation with 8 μM bp pBlueScript plasmid DNA. The positions of the Cy5‑labeled (indicated by asterisk) 40‑bp dsDNA and the strand‑exchange product are annotated alongside the gel images. Bottom: results from four independent experiments are plotted as the mean values ± s.e. e, ssDNA binding of RAD51 preincubated with excess of circular dsDNA in the presence of increasing concentrations (12.5, 25 and 50 nM) of WT and indicated X3C mutants. Results from three independent experiments are plotted as the mean values ± s.d. f, Strand exchange in the presence of AMP-PNP with increasing concentrations of RAD51 as indicated in c. RAD51 alone or RAD51 and WT or indicated X3C mutants mixed in a 2:1 ratio after preincubation with 8 μM bp pBlueScript plasmid DNA. Results from three independent experiments are plotted as the mean values ± s.e. Gel images in c,e,f were derived from the same experiment and processed in parallel.
