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.

Background & AimsRNase H2 is a holoenzyme, composed of 3 subunits (ribonuclease H2 subunits A, B, and C), that cleaves RNA:DNA hybrids and removes mis-incorporated ribonucleotides from genomic DNA through ribonucleotide excision repair. Ribonucleotide incorporation by eukaryotic DNA polymerases occurs during every round of genome duplication and produces the most frequent type of naturally occurring DNA lesion. We investigated whether intestinal epithelial proliferation requires RNase H2 function and whether RNase H2 activity is disrupted during intestinal carcinogenesis.MethodsWe generated mice with epithelial-specific deletion of ribonuclease H2 subunit B (H2bΔIEC) and mice that also had deletion of tumor-suppressor protein p53 (H2b/p53ΔIEC); we compared phenotypes with those of littermate H2bfl/fl or H2b/p53fl/fl (control) mice at young and old ages. Intestinal tissues were collected and analyzed by histology. We isolated epithelial cells, generated intestinal organoids, and performed RNA sequence analyses. Mutation signatures of spontaneous tumors from H2b/p53ΔIEC mice were characterized by exome sequencing. We collected colorectal tumor specimens from 467 patients, measured levels of ribonuclease H2 subunit B, and associated these with patient survival times and transcriptome data.ResultsThe H2bΔIEC mice had DNA damage to intestinal epithelial cells and proliferative exhaustion of the intestinal stem cell compartment compared with controls and H2b/p53ΔIEC mice. However, H2b/p53ΔIEC mice spontaneously developed small intestine and colon carcinomas. DNA from these tumors contained T>G base substitutions at GTG trinucleotides. Analyses of transcriptomes of human colorectal tumors associated lower levels of RNase H2 with shorter survival times.ConclusionsIn analyses of mice with disruption of the ribonuclease H2 subunit B gene and colorectal tumors from patients, we provide evidence that RNase H2 functions as a colorectal tumor suppressor. H2b/p53ΔIEC mice can be used to study the roles of RNase H2 in tissue-specific carcinogenesis.

Background: Tyr-226 and Tyr-390 in the ⌽29 DNA polymerase active site are implicated in the mechanism of translocation. Results: Y226F and Y390F differ in their effects on translocation and on dNTP and pyrophosphate binding. Conclusion: Mutations in the ⌽29 DNA polymerase and exonuclease active sites perturb dNTP or pyrophosphate binding rates. Significance: DNA polymerase architecture is finely tuned to integrate translocation and substrate binding.

To ensure the fidelity of replication, DNA polymerases preferentially incorporate nucleotide substrates complementary to a templating residue and select deoxyribonucleoside triphosphates (dNTPs) 3 rather than ribonucleoside triphosphates (rNTPs) in each catalytic cycle. This selection is achieved through a series of conformational transitions that precede the covalent step of phosphodiester bond formation ( Fig. 1) (1-5). One of these transitions is well characterized through the comparison of the crystal structures of polymerase-DNA complexes formed in the absence or presence of dNTP substrate complementary to the template residue at N ϭ 0 in the polymerase active site. These crystal structures reveal a major conformational difference between the two functional states. The polymerase domain has a conserved architecture that resembles a partially closed right hand (6, 7) comprising three subdomains. The palm subdomain contains residues required for the chemistry of catalysis, including the ligands for the two magnesium ions (metals A and B) that are essential for the reaction. The thumb subdomain positions the primer/template duplex in the active site, and the fingers subdomain contains residues essential for binding incoming nucleotide substrates. In complexes containing complementary dNTP, elements of the fingers subdomain rotate in toward the active site cleft to achieve a tight steric fit with the nascent base pair. Fluorescence resonance energy transfer (FRET) studies have shown for A family DNA polymerases that the transition between the open and closed states occurs rapidly in response to nucleotide binding (Fig. 1, Step 2.2) and is not rate-limiting for the catalytic cycle (2, 5). For the Klenow fragment of Escherichia coli DNA polymerase I (KF), stabilization of the fingers-closed state requires the metal ligand, Asp-882, presumably to form an essential contact with the Mg 2ϩ ion that is escorted into the closed complex with the incoming nucleotide substrate, but this step does not require the other metal ligand, .Pre-steady-state ensemble fluorescence and FRET experiments have revealed additional conformational changes that occur in response to nucleotide binding for KF (2, 4) (Fig. 1). After an initial rapid step that is reported by a change in the environment of the templating base at N ϭ 0 (Fig. 1, Step 2), a subsequent step that precedes fingers closing is reported as a change in the environment of the N ϭ ϩ1 template base (Fig. 1, Step 2.1). This step is promoted by dNTPs and rNTPs that are complementary to the templating base but not by non-* This work was supported, in whole or in part, by National Institutes of Health Grants 1RC2HG005553 from the NHGRI (to M. A.) and 1R01GM087484-01A2 from the NIGMS (to K. R. L. and M. A.). □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental