FIGURE 4.

DNA unwinding by mixed A257T-WT hexamers. A, WT gp4 (20 nm) was preincubated with various concentrations of A257T (10–100 nm) to make mixed hexamers, which were loaded on the fluorescein-labeled ds30 (23%) forked unwinding substrate with dTTP. The unwinding kinetics was measured by the stopped-flow fluorescence unwinding assay. B, the unwinding time traces show the decrease in amplitude with increasing fraction of A257T in the mixed hexamers. The kinetics was fit to the incomplete gamma function (29) to obtain the unwinding rates and amplitudes. C, the unwinding rate (filled circles) and amplitude (open circles) are plotted against a fraction of A257T.