Elementary steps in the DNA polymerase I reaction pathway

Abstract

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 kon = 1.2 X 10(6) M-1 s-1, koff = 0.25 s-1, and KD = 2 X 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 (kcat = 3.8 s-1, 22 degrees C) and dissociation of the enzyme from the template-primer under steady-state conditions (koffss = 0.1 s-1). The rapid quench time course of the polymerization reaction (kcat = 2.5 s-1, 20 degrees 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.

A Highly Sensitive 27 MHz Quartz-Crystal Microbalance as a Device for Kinetic Measurements of Molecular Recognition on DNA Strands

“…38 The smaller Kd = 5 -8 nM has been obtained in the combination of the short template similar to ours and the same Klenow fragment by using both the rapid quenching gel electrophoresis 39 and timeresolved fluorescent method. 40 Kinetic values obtained by our QCM methods were consistent with those obtained by the conventional methods.…”

Poliovirus RNA-dependent RNA Polymerase (3Dpol) Is Sufficient for Template Switchingin Vitro

“…Unlike the DNA-dependent DNA polymerases and reverse transcriptases (22,23,32,35), however, at equilibrium in the presence of excess primer/template all of the poliovirus polymerase in a reaction is not located at a primer/template junction. This conclusion is based on the observation that at least 30% of template nucleotides needed to be coated by primer to observe maximal rates of nucleotide incorporation (Fig.…”

“…2). In the case of the Klenow fragment of DNA polymerase I and human immunodeficiency virus reverse transcriptase, a stoichiometry of one 15-20-nt primer/400-1000-nt template can be used without observing any hysteretic effects on the kinetics of nucleotide incorporation (32,35).…”

Function and Structure Relationships in DNA Polymerases

“…With all of these approaches, however, there are caveats that preclude an unambiguous intetpretation of the results, so that our understanding of dNTP-binding remains at a rather primitive level. In the fIrst place, although a polymerase-dNTP binary complex can be formed, such a complex is not catalytically competent (72). This makes sense since the requirement for complementarity between the incoming dNTP and the DNA template dictates a substantial degree of contact between the nucleotide base and the template-primer.…”