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. 2016 Nov 28;45(2):749–761. doi: 10.1093/nar/gkw1125

Figure 3.

Figure 3.

Assembly of the RAD51 presynaptic complex. (A) Schematic showing the ssDNA curtain experiment for visualizing assembly of wild-type (dark) RAD51 using ssDNA-bound by hRPA-eGFP as a starting substrate. (B) Kymograph showing hRPA-eGFP stably bound to ssDNA in the absence of RAD51. (C) Kymograph showing displacement of hRPA-eGFP from the ssDNA upon injection of unlabeled RAD51 (indicated by black arrowhead) in the presence of 2 mM ATP, 1 mM Mg2+ and 5 mM Ca2+. The decrease in signal intensity is the result of hRPA-eGFP photobleaching, dissociation of hRPA-eGFP by RAD51 and the movement of molecules away from the TIRF field due to extension of the ssDNA. (D) The observed dissociation rates (see Supplementary Figure S3A), corrected to remove the contribution of photobleaching, are plotted against the RAD51 concentration. The constants are subsequently fitted to the hill equation to extract the Hill coefficient of 1.95 ± 0.322, Vmax = 0.011 ± 0.0042 s−1 and km = 828 ± 319 nM.