Figure 2.

Concentration dependence of the rate of dTTP incorporation opposite dA as the templating base with Mn²⁺. BST pol (1 μM) was pre-incubated with DNA_13A (80 nM) in reaction buffer and was mixed with increasing concentrations of dTTP [5, 10, 25, 50, 100, and 250 μM] containing 10 mM Mn²⁺. Reactions were quenched with 0.5 M EDTA (pH 8.0) at various times ranging from 4–200 ms. All data were obtained at 23 °C. (A) Gel images showing dTMP incorporation opposite dA at various [dTTP]; (B) plots of the amount of extended DNA product obtained as a function of time at various [dTTP]. Points are experimental, while curves are based on a fit of the data to Eq 1; (C) the single exponential rates obtained were plotted as a function of [dTTP] and fitted to Eq 2 to obtain a k_pol of 166 ± 14 s⁻¹ and a K_d,app of 22 ± 7 μM.