Figure 7.

PP_iinhibition on processive polymerization. T7 DNA polymerase E514Cou (120 nM), thioredoxin (4 μM), FAM-27/45 DNA (300 nM), BSA (0.1 mg/ml), and PP_i (0 or 8 mM) were mixed with dATP (100 μM), dTTP (100 μM), and Mg²⁺ (12.5 mM) to start the reaction. DNA lengths for each color are given at the top of the panel. The closed data points were collected without added PP_i, and the open data points had 8 mM PP_i in the reaction. The rates for the first and second incorporations for the simple model ED₂₇→ED₂₈→ED₂₉ for the reaction without PP_i were 182 ± 10 s⁻¹ and 41 ± 1 s⁻¹, respectively. The rates derived from the fit by simulation for the two incorporations for the reaction with 8 mM PP_i are 104 ± 8 s⁻¹ and 9.3 ± 0.6 s⁻¹, respectively. Lines through the data were computed based on global data fitting shown in Figure 8.