Nanopores can be used to analyse DNA by monitoring ion currents as individual strands are captured and driven through the pore in single file order by an applied voltage. Here we show that serial replication of individual DNA templates can be achieved by DNA polymerases held at the α-hemolysin nanopore orifice. Replication is blocked in the bulk phase, and is initiated only after the DNA is captured by the nanopore. We used this method, in concert with active voltage control, to observe DNA replication catalyzed by bacteriophage T7 DNA polymerase (T7DNAP) and by the Klenow fragment of DNA polymerase I (KF). T7DNAP advanced on a DNA template against an 80 mV load applied across the nanopore, and single nucleotide additions were measured on the millisecond time scale for hundreds of individual DNA molecules in series. Replication by KF was not observed when this enzyme was held atop the nanopore orifice at 80 mV applied potential. Sequential nucleotide additions by KF were observed upon controlled voltage reversals.

Background: DNA polymerases translocate along DNA by one nucleotide in each catalytic cycle. Results: The DNA polymerase translocation step is observed with single nucleotide and submillisecond precision. Conclusion: DNA polymerase complexes fluctuate between pre-and post-translocation states and are rectified to the posttranslocation state by dNTP. Significance: These results provide insight into the translocation mechanism and its integration into the DNA polymerase catalytic pathway.

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.

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.

Exonucleolytic editing of incorrectly incorporated nucleotides by replicative DNA polymerases (DNAPs) plays an essential role in the fidelity of DNA replication. Editing requires that the primer strand of the DNA substrate be transferred between the DNAP polymerase and exonuclease sites, separated by a distance that is typically on the order of ∼30 Å. Dynamic transitions between functional states can be quantified with single-nucleotide spatial precision and submillisecond temporal resolution from ionic current time traces recorded when individual DNAP complexes are held atop a nanoscale pore in an electric field. In this study, we have exploited this capability to determine the kinetic relationship between the translocation step and primer strand transfer between the polymerase and exonuclease sites in complexes formed between the replicative DNAP from bacteriophage Φ29 and DNA. We demonstrate that the pathway for primer strand transfer from the polymerase to exonuclease site initiates prior to the translocation step, while complexes are in the pre-translocation state. We developed a mathematical method to determine simultaneously the forward and reverse translocation rates and the rates of primer strand transfer in both directions between the polymerase and the exonuclease sites, and we have applied it to determine these rates for Φ29 DNAP complexes formed with a DNA substrate bearing a fully complementary primer–template duplex. This work provides a framework that will be extended to determine the kinetic mechanisms by which incorporation of noncomplementary nucleotides promotes primer strand transfer from the polymerase site to the exonuclease site.