Supplementary Figure 4: The RPA fragment comprised of FAB domains displays rapid and monophasic binding to ssDNA and single-molecule analysis of RPA-FAB-AMB543-ssDNA interaction reveal the presence of two distinct fluorescence states and more rapid dissociation than trimeric RPA-DBD-AMB543.
From: Dynamics and selective remodeling of the DNA-binding domains of RPA
The FAB region of RPA (DBDs F, A and B) was purified and labeled with MB543 at DBD-A (RPA-FAB-AMB543), and (a) produces a robust change in fluorescence upon binding to ssDNA. (b) Stopped flow analysis shows rapid binding of RPA-FAB-A MB543 (100 nM) to 100 nM of ssDNA of increasing lengths (dT)n. A minimum of 15 nt is required to observe binding. (c) RPA-FAB-Af binding dynamics on ssDNA were measured by monitoring the change in fluorescence upon binding to increasing concentrations of [(dT)30] ssDNA. (d) Measurement of DNA binding kinetics reveal kon (1.1±0.1 × 108 M-1s-1). The RPA-FAB-AMB543 binding data were fit to a single exponential plus linear equation to obtain kobs,1. (e) Experimental scheme for visualization of the binding and conformational dynamics of FAB. Binding of the fluorescently-labeled FAB (1.0, 0.75, 0.50 µM) to ssDNA (blue line) tethered to the surface of TIRFM flow cell (grey line) brings the MB543 fluorophore within the evanescent field and its excitation. NA – neutravidin, b – biotin. (f) Representative fluorescence trajectories depicting binding (appearance and disappearance of the signal) and conformational dynamics (change in fluorescence without FAB dissociation) of the individual RPA-FAB-AMB543 molecules labeled within the DBD-A. Purple lines represent normalized fluorescence. Black lines represent the results of ebFRET fitting of the experimental data to the three-state model (where state 1 is the off state, while states 2 and 3 are the two bound states with different fluorescent intensities). The levels for the respective states are indicated by dashed lines. The top trajectory is representative of the most commonly observed type with short binding events and rare transitions between fluorescence states. At each RPA-FAB-AMB543 concentration, we also observed several trajectories displaying long binding events as the bottom trajectory here. (g) The dissociation rate constant, koff was determined from the decay rate of the on state dwell time (when trajectories were fitted with the two state model). The average at the three concentrations is shown with standard deviation. (h) The association rate, Von was determined as the number of binding events observed in each experiment divided by the product of the observation time and the number of trajectories observed at 1 nM RPA-FAB-AMB543. The association rate constant, kon was determined by calculating the slope of the Von dependence on RPA-FAB-AMB543 concentration. The equilibrium dissociation rate constant, Kd is the ratio of the two rate constants. All calculations accounted for the RPA-FAB-AMB543 labeling efficiency.
