FIGURE 5.
Biphasic kinetics of correct dNTP incorporation in the presence of a DNA trap. A preincubated solution of Dpo4 (120 nm) and 5′-32P-labeled 20/26-mer-dGAP (▪, 30 nm) or 21/26-mer-dGAP (•, 30 nm) was mixed rapidly with 21/41-mer D-1 (5 μm) and dCTP (▪, 1.2 mm) or dGTP (•, 1.2 mm). The reaction was quenched with 0.37 m EDTA after various times. The product concentration was plotted as a function of reaction time for each DNA substrate which was then fit to Equation 4. For 20/26-mer-dGAP, the fast phase had a reaction amplitude of 2.0 ± 0.7 nm and a reaction rate of 11 ± 6 s-1, whereas the slow phase had a reaction amplitude of 18 ± 1 nm and a reaction rate of 0.7 ± 0.1 s-1. For 21/26-mer-dGAP, the fast phase had a reaction amplitude of 0.9 ± 0.1 nm and a reaction rate of 1.9 ± 0.3 s-1, whereas the slow phase had a reaction amplitude of 5.7 ± 0.2 nm and a reaction rate of 0.031 ± 0.003 s-1.