FIGURE 6.
Fluorescence-based DNA binding. A schematic representation of the RFC subunit arrangement and a schematic of the assay are shown to the right of the data. Equilibrium DNA binding is shown for WT RFC (black), WT RFC without ATPγS (gray), Rfc1GAT (purple), Rfc2GAT (green), Rfc3GAT (red), and Rfc4 GAT (blue). DNA-DCC fluorescence was measured in solutions containing 10 nm DNA-DCC, 0.5 mm ATPγS, and 0–2530 nm clamp loader in assay buffer. The average relative DCC intensities and standard deviations from three independent experiments are plotted and fit to Equation 3. Dissociation constants (Kd values) were calculated for each experiment with WT RFC, and the average values and standard deviations are given in the text. Final assay buffer contained 30 mm HEPES, pH 7.5, 150 mm NaCl, 2 mm DTT, 10 mm MgCl2, 10% glycerol, and 750 mm maltose. Error bars represent S.D.