Figure 1.
Pre-steady-state and steady-state kinetics of correct dTTP incorporation by hPolε exo−. A, the burst kinetics of dTTP incorporation were measured by mixing a preincubated solution of hPolε exo− (20 nm, UV concentration) and 5′-radiolabeled D-1 DNA substrate (80 nm) with 100 μm dTTP and Mg2+ and then quenching at various time intervals with addition of EDTA. The data were fit to Equation 1 to yield a fast exponential rate constant of 139 ± 27 s−1, a slow exponential rate constant of 1.3 ± 0.5 s−1, and a linear phase rate constant of 0.0044 ± 0.0008 s−1. B, the steady-state DNA polymerization rate constant was measured by mixing a preincubated solution of hPolε exo− (1 nm, active site concentration) and 5′-radiolabeled D-1 DNA substrate (400 nm) with dTTP (100 μm) and Mg2+ for varying reaction times and then quenching the reaction with the addition of EDTA. Product concentration was plotted against time and the data were fit to Equation 2 to yield a kss of 0.0070 ± 0.0003 s−1. C, to directly measure the DNA dissociation rate constant, k-1, a preincubated solution of hPolε exo− (50 nm, UV concentration) and 5′-radiolabeled D-1 DNA substrate (100 nm) was mixed with unlabeled D-1 DNA substrate (2.5 μm) for varying incubation times before the reaction was initiated with the addition of dTTP (100 μm) and Mg2+. The reaction was allowed to proceed for 15 s and then quenched with the addition of EDTA. Product concentration was plotted against time and the data were fit to Equation 3 to yield a k-1 of 0.0058 ± 0.0007 s−1.