Fig. 3.

Incorrect and correct forward and reverse reactions with D473G Pfu pol. At the top of each plot, a sketch is shown to describe the reaction. All plots are shown as percentage of 22mer vs. time. (A) Incorrect incorporation of dGTP opposite T, at various dGTP concentrations: 200 μM (black circles), 400 μM (red circles), 600 μM (green triangles), 700 μM (yellow triangles), and 800 μM (blue squares). Reactions in the presence of 2.5 mM PP_i were carried out for 4 h. (B) Pyrophosphorolysis reaction starting with the incorrectly matched [DNA₂₂ (G•T)] at various dGTP concentrations, in the presence of 5 mM PP_i: 25 μM (black circles), 50 μM (red circles), and 100 μM (green triangles). Pyrophosphorolysis is the primary reaction occurring initially, followed by incorporation until a steady state is reached. Very little DNA₂₁ (G•C) results from this reverse reaction (note scale on y axis). (C) Correct incorporation of dATP by D473G Pfu pol at various dATP concentrations: 156 nM (black circles), 312 nM (red circles), 625 nM (green triangles), 1,250 nM (yellow triangles), and 2,500 nM (blue squares). In all cases, dGTP is present at 50 μM in the reaction to prevent pyrophosphorolysis continuing beyond the 20mer primer. Reactions were carried out for 4 h. (D) Pyrophosphorolysis reaction starting from DNA₂₂ (A•T) at various dATP concentrations: 156 nM (black circles), 312 nM (red circles), 625 nM (green triangles), 1,250 nM (yellow triangles), and 2,500 nM (blue squares). Also, dGTP is present at 50 μM in the reaction to prevent pyrophosphorolysis continuing beyond the 20mer primer. Reactions were carried out for 8 h. Reactions with D473G Pfu pol apparently begin with a large decrease in the amount of initial 22mer, and then there is a slow increase again with incorporation taking over for pyrophosphorolysis until the reactions reach a steady state.