RPA-eGFP can be rapidly replaced from ssDNA by Rad51.

<p>(A) Schematic illustrating the predicted outcome for an ssDNA curtain experiment (side view) where RPA-eGFP is replaced by unlabeled Rad51. The loss of fluorescence as RPA-eGFP is displaced by Rad51 also coincides with an increase in the length of the ssDNA, which causes an increase in the transverse fluctuations of the ssDNA molecules. (B) The upper panel shows a kymograph of RPA-eGFP bound to ssDNA over time in the absence of Rad51, and the middle panel shows how RPA-eGFP is rapidly displaced from the ssDNA upon injection of 750 nM unlabeled Rad51 with 2.5 mM ATP. Also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087922#pone.0087922.s002" target="_blank">Video S2</a>. The lower panel shows an example of a single-tethered ssDNA molecule, which illustrates how Rad51 binding coincides with displacement of RPA-eGFP and extension of the ssDNA. This single-tethered measurement was made using 650 nM Rad51 and 1 mM ATP. (C) RPA-eGFP signal versus time collected at different concentrations of Rad51 (as indicated) in the presence of 2.5 mM ATP in buffer containing 50 mM KCl. Each curve represents the normalized average calculated from 11 to 70 different ssDNA molecules. Shaded regions correspond to the standard deviation for each data set. The data were fit to single exponential decays (solid lines), and loss of signal reflects a combination of photo-bleaching (as reflected in the minus Rad51 control), Rad51-induced dissociation of RPA-eGFP, and corresponding extension of the ssDNA, which causes the time-averaged position of the molecules to move further away from the surface.