Figure 2: Effects of gamma-BCD mutants. (a) Relative steady-state fluorescence emission spectra obtained with excitation at 460 nm. 500 nM fluorescein attached to D106C delta-A in clamp loader + 4 uM D253C beta labeled with TMR. A schematic representation of the experiment is shown, and the individual spectra are labeled to show the components present. (b) Clamp binding and release as monitored by the reduction in donor fluorescence at 515 nm. The upper panel shows clamp binding (red bars) as calculated by Fbind-mutant/Fbind-wt where Fbind = Fno ATP - FATP (compare black and red traces in panel a). The lower panel shows clamp release (green bars for 6.6 uM DNA, blue bars for 5 mM ADP plus DNA) calculated by (Frelease-mutant/Fbind-mutant )/(Frelease-wt/Fbind-wt) where Fbind is as described above and Frelease = Frelease - FATP (compare green traces for DNA-dependent release or blue traces for ADP+DNA-dependent release in panel a). (c) ATPase assays. Data plotted is the stimulation in ATPase rates from addition of primer-template DNA where 1-fold is equivalent to no stimulation. Error bars represent the standard error of at least two measurements. The relative basal ATPase rates (in the absence of DNA) are indicated below the graph.