Figure 3.

Pre-steady-state kinetics of correct nucleotide incorporation. A, a preincubated solution of hPolε exo− (100 nm, UV concentration) and 5′-radiolabeled D-1 DNA substrate (20 nm) was rapidly mixed with 1.25 (purple), 2.5 (blue), 5 (green), 10 (yellow), 80 (orange), or 200 μm (red) dTTP and Mg²⁺ for varying incubation times before the reaction was quenched with the addition of EDTA. Product concentration was plotted against time and the data were fit to Equation 5. The inset shows the first 0.2 s of each time course. B, the k_fast and k_slow values for each dTTP concentration were plotted against their respective dTTP concentration. The plot of k_fast versus [dTTP] was fit to Equation 7 to yield a k_max of 411 ± 26 s⁻¹ and a $K_d^{dTTP}$ of 11 ± 2 μm. The plot of k_slow versus [dTTP] was fit with a smoothed line. C, the A_fast and A_slow values for each dTTP concentration were plotted against their respective dTTP concentration, and the data were fit with a smoothed line.