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Complexes of phi29 DNA polymerase and DNA fluctuate on the millisecond time scale between two ionic current amplitude states when captured atop the α-hemolysin nanopore in an applied field. The lower amplitude state is stabilized by complementary dNTP and thus corresponds to complexes in the post-translocation state. We have demonstrated that in the upper amplitude state, the DNA is displaced by a distance of one nucleotide from the post-translocation state. We propose that the upper amplitude state corresponds to complexes in the pre-translocation state. Force exerted on the template strand biases the complexes toward the pre-translocation state. Based on the results of voltage and dNTP titrations, we concluded through mathematical modeling that complementary dNTP binds only to the post-translocation state, and we estimated the binding affinity. The equilibrium between the two states is influenced by active site-proximal DNA sequences. Consistent with the assignment of the upper amplitude state as the pre-translocation state, a DNA substrate that favors the pre-translocation state in complexes on the nanopore is a superior substrate in bulk phase for pyrophosphorolysis. There is also a correlation between DNA sequences that bias complexes toward the pre-translocation state and the rate of exonucleolysis in bulk phase, suggesting that during DNA synthesis the pathway for transfer of the primer strand from the polymerase to exonuclease active site initiates in the pre-translocation state.

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. [ABSTRACT FROM AUTHOR]