Figure 3. Single-molecule analysis quantifies the conformational dynamics of DBDs and the effect of the recombination mediator Rad52.

a & b) Experimental scheme for visualizing conformational dynamics of DBD-A and DBD-D. Binding of fluorescently labeled RPA to a surface-tethered ssDNA (purple line) brings the MB543 fluorophore within the evanescent field of TIRFM. NA – neutravidin, b – biotin. c & d) Representative fluorescence trajectories for individual RPA-DBD-A^MB543and RPA-DBD-D^MB543 molecules, (purple and green lines, respectively). Black lines are the results of ebFRET fitting. Additional examples of representative trajectories are presented in Supplementary Datasets 2 & 3. e & f) Experimental scheme for visualizing the effect of Rad52 on the conformational dynamics of DBD-A and DBD-D. g & h) Representative fluorescence trajectories depicting conformational dynamics of the individual RPA-DBD-A^MB543and RPA-DBD-D^MB543 molecules upon addition of Rad52. Additional trajectories are shown in the Supplementary Datasets 5 & 6. i-j) Dwell time histograms for the four fluorescent states obtained by the ebFRET fitting of RPA-DBD-A^MB543 trajectories from three independent experiments after buffer wash (i), and Rad52 wash (j). The trajectories were reduced from 120 sec to 210 sec. Solid lines represent single-exponential fit. The data are summarized in Supplementary Table 1. k) Fractional visitation to each state available to RPA-DBD-A^MB543 alone (grey) and in the presence of Rad52 (blue). The 2-way ANOVA analysis suggests no significant differences between the visitation frequencies in the presence and absence of Rad52 (p>0.1). l) Stability of each state available to RPA-DBD-A^MB543 alone (grey) and in the presence of Rad52 (blue). The data on Y axis are the lifetimes for the respective dwell time distributions. m - p) The same analysis was carried out for RPA-DBD-D^MB543. Only three fluorescence states were detected in the presence of Rad52. NP – not present. Statistical analysis is performed by ANOVA (***, and **** correspond to p=0.0001 and p<0.0001, respectively).