Kinetic Mechanism of Translocation and dNTP Binding in Individual DNA Polymerase Complexes

Abstract

Complexes formed between phi29 DNA polymerase (DNAP) and DNA fluctuate discretely between the pre-translocation and post-translocation states on the millisecond time scale. The translocation fluctuations can be observed in ionic current traces when individual complexes are captured atop the α-hemolysin nanopore in an electric field. The presence of complementary 2′-deoxynucleoside triphosphate (dNTP) shifts the equilibrium across the translocation step toward the post-translocation state. Here we have determined quantitatively the kinetic relationship between the phi29 DNAP translocation step and dNTP binding. We demonstrate that dNTP binds to phi29 DNAP-DNA complexes only after the transition from the pre-translocation state to the post-translocation state; dNTP binding rectifies the translocation but it does not directly drive the translocation. Based on the measured time traces of current amplitude, we developed a method for determining the forward and reverse translocation rates, and the dNTP association and dissociation rates, individually at each dNTP concentration and each voltage. The translocation rates, and their response to force, match those determined for phi29 DNAP-DNA binary complexes and are unaffected by dNTP. The dNTP association and dissociation rates do not vary as a function of voltage, indicating that force does not distort the polymerase active site, and that dNTP binding does not directly involve a displacement in the translocation direction. This combined experimental and theoretical approach, and the results obtained, provide a framework for separately evaluating the effects of biological variables on the translocation transitions and their effects on dNTP binding.

Modulation of DNA Polymerase Noncovalent Kinetic Transitions by Divalent Cations

“…Specifically, we showed that the forward translocation precedes dNTP binding; the translocation fluctuation is rectified but not driven by dNTP binding, and dNTP has no influence on the rates of the translocation fluctuations (Fig. 2B) (25). Furthermore, we established that transfer of the primer strand from the polymerase to the exonuclease site occurs prior to the forward translocation fluctuation; the pre-translocation state is therefore the branch point between the DNA synthesis and editing pathways (Fig.…”

“…Addition of dNTP complementary to the templating base at n ϭ 0 (dGTP) stabilizes the lower amplitude post-translocation state ( Fig. 2A, ii) (23,25). In contrast to complexes formed with DNA1-H_H ( Fig.…”

“…1, A and B) that permits quantification of the rates of translocation fluctuations, rates of the primer strand transfer between the polymerase and exonuclease sites, and rates of dNTP binding, in individual DNAP-DNA complexes (23)(24)(25)(26). We used the B-family replicative ⌽29 DNAP, which serves as an excellent model system for leading strand DNA synthesis catalyzed in more complex B family replisomes.…”

“…We showed that the periods of rapid fluctuation are due to transitions between the pre-translocation and post-translocation states and that the pauses in the upper amplitude arise when the primer strand is transferred from the polymerase active site to the exonuclease site (26). Thus, the upper amplitude comprises the pre-translocation state in the polymerase site and the state in which the primer strand occupies the exonuclease site; the lower amplitude comprises the post-translocation state in the polymerase site (23)(24)(25)(26).…”

“…This applied force shifts the translocation equilibrium toward the upper amplitude, pre-translocation state (23,24); it impedes the rate of the forward translocation and increases the rate of the reverse translocation (23,24), but it does not affect the rates of dNTP binding (25) or the rates of primer strand transfer between the polymerase and exonuclease sites (26). Based on translocation fluctuations measured under varying opposing force loads and in the presence of varying nucleotide concentrations, we established the kinetic relationship of the translocation to the step of nucleotide binding and to the step of primer strand transfer between the polymerase and exonuclease sites (Fig.…”