Elementary Steps in the DNA Polymerase I Reaction Pathway

The polymerization reaction catalyzed by Escherichia coli DNA polymerase I (Pol I) has been studied by using the homopolymer template-primer system poly(dA)· oligo(dT). Isotope-partitioning experiments indicate that the reaction follows an ordered mechanism in which Pol I first combines with template-primer to form an E·poly complex followed by addition of MgTTP and catalysis. The parameters governing the binding of Pol I to the template-primer are k_on = 1.2 × 10⁶ M⁻¹ s⁻¹, k_off = 0.25 s⁻¹, and K_D = 2 × 10⁻⁷ M. Efforts to demonstrate the catalytic competence of the binary E·MgTTP complex were unsuccessful. Following initiation of the catalytic cycle, Pol I catalyzes the incorporation of an average of 40–50 TTP molecules into polymer before dissociating from the template-primer. The processive nature of the polymerization reaction as reflected by the isotope-trapping time dependence can be accounted for by a model in which processive synthesis is treated as a simple partitioning between continued polymerization (k_cat = 3.8 s⁻¹, 22 °C) and dissociation of the enzyme from the template-primer under steady-state conditions (k_off^ss = 0.1 s⁻¹). The rapid quench time course of the polymerization reaction (k_cat = 2.5 s⁻¹, 20 °C) exhibited a pre-steady-state burst consistent with two partially rate-determining steps, one of which precedes the actual chemical phosphodiester bond-forming step (k = 4.6 s⁻¹) and the other which follows this step (k = 4.0 s⁻¹). Binding of MgTTP to the E·poly complex was shown to be a rapid equilibrium step by steady-state isotope-partitioning experiments. This suggested that the first rate-determining step may be a first-order isomerization which follows the binding of substrates and precedes bond formation.