Elementary Steps in the DNA Polymerase I Reaction Pathway

F. R. Bryant, K. A. Johnson, Stephen Benkovic

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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 × 106 M−1 s−1, koff = 0.25 s−1, and KD = 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 (kcat = 3.8 s−1, 22 °C) and dissociation of the enzyme from the template-primer under steady-state conditions (koff ss = 0.1 s−1). The rapid quench time course of the polymerization reaction (kcat = 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.

Original languageEnglish (US)
Pages (from-to)3537-3546
Number of pages10
JournalBiochemistry
Volume22
Issue number15
DOIs
Publication statusPublished - Jan 1 1983

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All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Bryant, F. R., Johnson, K. A., & Benkovic, S. (1983). Elementary Steps in the DNA Polymerase I Reaction Pathway. Biochemistry, 22(15), 3537-3546. https://doi.org/10.1021/bi00284a001