Dynamics of the Translocation Step Measured in Individual DNA Polymerase Complexes

Abstract

Complexes formed between the bacteriophage phi29 DNA polymerase (DNAP) and DNA fluctuate between the pre-translocation and post-translocation states on the millisecond time scale. These fluctuations can be directly observed with single-nucleotide precision in real-time ionic current traces when individual complexes are captured atop the alpha-hemolysin nanopore in an applied electric field. We recently quantified the equilibrium across the translocation step as a function of applied force (voltage), active-site proximal DNA sequences, and the binding of complementary dNTP. To gain insight into the mechanism of this step in the DNAP catalytic cycle, in this study, we have examined the stochastic dynamics of the translocation step. The survival probability of complexes in each of the two states decayed at a single exponential rate, indicating that the observed fluctuations are between two discrete states. We used a robust mathematical formulation based on the auto-correlation function to extract the forward and reverse rates of the transitions between the pre-translocation state and the post-translocation state from ionic current traces of captured phi29 DNAP-DNA binary complexes. We evaluated each transition rate as a function of applied voltage to examine the energy landscape of the phi29 DNAP translocation step. The analysis reveals that active-site proximal DNA sequences influence the depth of the pre-translocation and post-translocation state energy wells and affect the location of the transition state along the direction of the translocation.

Modulation of DNA Polymerase Noncovalent Kinetic Transitions by Divalent Cations

“…In addition, as we have previously reported, when complexes formed between either the wild type or D12A/D66A enzyme and the DNA1-H_H substrate are captured in the presence of high [Me 2ϩ ], plots of survival probability versus dwell time of the upper amplitude are fit by a single exponential decay function. This decay rate corresponds to the inverse of the forward translocation rate (24,26). Thus, for the DNA1-H_H complexes at high [Me 2ϩ ], a population of dwell time samples that corresponds to stays of the primer strand in the exonuclease site cannot be resolved.…”

“…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.…”

“…In prior studies (23,24,26,32,33), we applied these capabilities to examine the kinetic mechanisms by which mutations of highly conserved DNAP residues, alterations in DNA substrate Complexes were formed between the D12A/D66A mutant of ⌽29 DNAP and (i) DNA1-H_OH; or (ii) and (iii) DNA1-H_H. In (iii) 4 M dGTP was present in the cis chamber.…”