Fig. 1.

Forward and reverse reactions for misincorporation of dGTP opposite T by Vent pol. (A) Gel data obtained for a representative reaction with 1.6 mM dGTP and 11 mM PP_i. The sketch at the top illustrates the reaction proceeding from primer 21mer to 22mer, by incorporation of dGTP opposite the underlined templating T, but the reverse reaction is not detected. The amount of DNA₂₂ (G•T) increases with time. At the end of 48 h incubation, 1 mM dATP is added and allowed to react for 20 min (++dATP) to show the enzyme is still active at the end of reaction. Note that DNA₂₂ (G•T) and DNA₂₂ (A•T) are clearly separated as bands in the gel. The final two lanes show two controls, one containing only dGTP (−PP_i) and one only PP_i (−dNTP), demonstrating that the enzyme can fully extend the 21mer to 22mer with dGTP and no PP_i and can perform extensive pyrophosphorolysis with PP_i and no dNTP, respectively. (B) Amount of extended primer (% 22mer) as a function of reaction time, at various dGTP concentrations: 100 μM (black circles), 200 μM (red circles), 400 μM (green triangles), 800 μM (yellow triangles), and 1,600 μM (blue squares). By the end of 48-h reactions, the amount of extended primer DNA₂₂ (G•T) is still increasing slightly. (C) Starting from DNA₂₂ (G•T), Vent pol was unable to perform pyrophosphorolysis. The first lane shows the starting 22mer before any reaction (t = 0). The subsequent lanes are quenched at t = 12 h, 24 h, and 48 h incubation with 200 μM dGTP and 11 mM PP_i. (C, Left) The lane marked −dGTP shows that no pyrophosphorolysis is occurring without the inclusion of dGTP in reaction mixture, or at higher temperature (72 °C), or with inclusion of dATP in the reaction mixture (+dATP), or with excess enzyme (++pol). (C, Right) After 48 h, 100 nM of DNA₂₁ (G•C) was added to reveal the enzyme was still active (lane 1), and then an aliquot was quenched 16 h later (lane 2).