Crystal structure of the active site of phage T7 DNA polymerase (PDB entry 1T8E). A, T7 DNA polymerase in complex with thioredoxin, a primer-template, and a dNTP in the active site. The two Mg²⁺ ions are shown in green. B, enlargement of the active site of T7 DNA polymerase containing the 3′ terminus of the primer with the two Mg²⁺ ions (green) and dATP (left) or PP_i (right) from an overlay of the two metal ions in the Dpo4 crystal structure together with PP_i (PDB entry 2ago). The structural alignment and figure preparation were created using PyMOL. The two Mg²⁺ ions are coordinated through the protein side chain moieties and the phosphate backbone of the DNA and NTP or PP_i.

Pyrophosphorolysis and pyrophosphate exchange. A, gel analysis of pyrophosphorolysis catalyzed by T7 DNA polymerase. The reaction contained 100 nm T7 DNA polymerase deficient in the 3′–5′-exonuclease activity, 5 nm [5′-³²P]DNA primer (21-mer) annealed to template (26-mer), 40 mm Tris-HCl (pH 7.5), 10 mm MgCl₂, 10 mm DTT, 50 mm potassium glutamate, 250 μm PP_i, and 250 μm dTTP where indicated. After incubation at 37 °C for 20 min, the radioactive products were analyzed by electrophoresis through a 25% polyacrylamide gel containing 3 m urea and visualized using autoradiography. 1st lane, 5′-³²P-labeled primer alone (no template); 2nd lane, primer-template and no PP_i; 3rd lane, primer-template and PP_i; 4th lane, primer-template, PP_i, and dTTP. B, nucleotide sequences of the various DNA primers annealed to the same template. The primers are designated as follows: recessed, mispair-3, mispair-6, bubble (three mismatched bases), and blunt as indicated. C, effect of primer-template configuration on pyrophosphorolysis. The five primers described in B were incubated with T7 DNA polymerase and increasing amounts of PP_i as presented above. The products of the reaction were analyzed by electrophoresis through a 18% polyacrylamide gel containing 7 m urea and visualized using autoradiography. D, quantification of pyrophosphorolysis. The bands in the gels presented in C were analyzed using autoradiography. E, ratio of pyrophosphorolysis to exonuclease activity. The total intensities of the products in lane 1 (no PP_i) and lane 4 were quantified by integration of gel tracks the same as in D. The ratios between these lanes were calculated and represent the ratio of pyrophosphorolysis to exonuclease activity for each primer-template configuration.

Pyrophosphorolysis and PP_i exchange catalyzed by T7 DNA polymerase in bulk and at the single molecule level. A, each pyrophosphorolysis reaction was performed as in Fig. 2B using the recessed primer annealed to the DNA template with the following modifications: same amount of [³²P]PP_i and varying amounts (0, 0.25, 1, and 4 mm) of PP_i in the absence or presence of 5 mm dTTP to induce pyrophosphorolysis or PP_i exchange. After incubation for 10 min at 37 °C 0.4 μl of the reaction mixture was spotted onto polyethyleneimine cellulose TLC plate and developed using 0.5 m LiCl, 0.5 m sodium formate. To better separate the [³²P]PP_i used in the reaction from the accumulated product ([³²P]dNTP), we coupled the reaction to pyrophosphatase (0.1 units). The radioactive products (dTTP) were visualized using autoradiography. B, graphs represent quantification of dNPPP formation from the pyrophosphorolysis reaction when increasing amounts of unlabeled PP_i are present. A full view on TLC is available in supplemental Fig. 3A, top panel. C, experimental design for the single molecule analysis of leading strand synthesis mediated by T7 DNA polymerase and gp4 helicase in the presence or absence of pyrophosphate. Duplex λ DNA (48.5 kb) is attached to the surface of the flow cell via the 5′-end of the fork using biotin-streptavidin interaction, and the 3′-end is attached to a paramagnetic bead using digoxigenin-anti-digoxigenin interaction. The replication reaction in the flow cell contains 50 nm gp4 (hexameric concentration), 100 nm T7 DNA polymerase in buffer consisting of 600 μm each of dATP, dTTP, dCTP, and dGTP, 10 mm DTT, 10 mm MgCl₂, and 1 mm pyrophosphate if indicated. DNA synthesis by proteins leads to conversion of the dsDNA to ssDNA, resulting in shortening of the DNA ligand that was accompanied by the movement of the bead against the direction of the flow. D, rate and processivity of leading strand synthesis by T7 DNA polymerase and gp4 helicase in the presence or absence of pyrophosphate. Examples of single molecule trajectories for leading strand synthesis are shown. Rate and processivity were calculated by fitting the distributions of individual single molecule trajectories using Gaussian and exponential decay distributions, respectively. Rate and processivity were calculated by fitting the distributions of individual single molecule trajectories using Gaussian and exponential decay distributions, respectively (fitted histograms shown are for gp5/trx and gp4 in the presence and the absence of 1 mm PP_i). Thirty three single events were used to calculate rate and processivity of the leading strand synthesis in the presence and the absence of pyrophosphate. Standard errors represent the accuracy in fitting of these distributions.

Compounds that mimic PP_i. A, compounds (triphosphate, VV, phosphonoformate, and phosphonoacetate) were selected on the basis of their charge, size, and geometry of the oxygen that connects the two acyl groups of PP_i. B, each of the compounds (250 μm) depicted in A were examined for their ability to mediate a pyrophosphorolysis-like reaction in the presence of 10 mm MgCl₂ or 1 mm MnCl₂, using the same primer-template DNA and reaction conditions as in Fig. 2A. C, effect of PP_i or VV on pyrophosphorolysis or pyrovanadolysis upon substitution of Mn²⁺ for Mg²⁺. The reaction contained 5 mm PP_i or VV and increasing amounts of MgCl₂ or MnCl₂ (0, 0.25, 1, and 2 mm) (left panel) or 10 mm MgCl₂, 1 mm MnCl₂ and increasing amounts of PP_i or VV (0, 0.25, 1, and 2 mm) (right panel). The primer-template used consisted of the radiolabeled recessed primer as described in Fig. 2B. The reaction conditions and gel analysis of the products in B and C were otherwise as described in Fig. 2.

Identification of products of pyrophosphorolysis and pyrovanadolysis. A, schematic representation of the DNA substrate with the 3′-labeled primer strand. The substrate was prepared by using T7 DNA polymerase (T7DNAP) to add [³²P]dGMP to the 3′ terminus of the primer strand as described under “Experimental Procedures.” B, pyrophosphorolysis and pyrovanadolysis were catalyzed by T7 DNA polymerase using 5 mm PP_i or VV, respectively, in a reaction containing the radioactively labeled DNA depicted in A. MgCl₂ (10 mm) was used in the reaction containing PP_i and MnCl₂ (1 mm) in the reaction containing VV. Lane 1 contains only [α-³²P]dTTP as a marker. Lane 2, only DNA substrate. Lane 3, reaction mixture without PP_i or VV containing 10 mm MgCl₂ and T7 DNA polymerase (wild type). Lane 4 contains reaction mixture with genetically modified T7 DNA polymerase (gp5-D5D65/trx), MgCl₂, and PP_i. Lane 5 contains reaction mixture with genetically modified T7 DNA polymerase, MnCl₂, and VV. The products of exonuclease activity, pyrophosphorolysis, and pyrovanadolysis were visualized using TLC and developed in 0.5 m LiCl and 2 n acetic acid and analyzed using autoradiography.

Distance distribution of PP_i, VV, and protein ligands around the manganese in the active site of T7 DNA polymerase. A, EXAFS fitting results of T7 DNA polymerase. Manganese K-edge EXAFS data (inset) and its Fourier transform in real space for PP_i (top) or VV (bottom) bound in the active site of T7 DNA polymerase. Best fits (cyan) to the experimental data (black) are indicated. EXAFS analysis was performed using the iFFEFIT analysis package. The theoretical EXAFS signal was constructed of the bacteriophage T7 DNA polymerase (PDB entry 1T8E) and calculated using FEFF embedded in the iFFEFIT package (24, 25). B, columns representing the Mn-O, Mn-P, and Mn-V coordination bond distances based on EXAFS fitting results. The fitting parameters are summarized in Table 1. C, schematic representation of T7 DNA polymerase active site configuration when bound to Mn- PP_i and Mn-VV, respectively. Bond distances were obtained from EXAFS results summarized in Table 1 (black). The distances of terminal and bridging oxygens in phosphorus or vanadium and the angle of bridging oxygen (POP and VOV) is based on available crystal structures of PP_i and VV free or bound to an enzyme active site (summarized in supplemental Table 1).

Molecular model of pyrophosphorolysis and pyrovanadolysis. A, proposed mechanism for pyrophosphorolysis (left panel) and pyrovanadolysis (right panel). In pyrophosphorolysis PP_i attacks the phosphate backbone of the 3′-end of the primer to yield dNTP. A water molecule may stabilize the 3′-end of the primer and make it a better leaving group. In pyrovanadolysis the formation of dNMPVV is transient and is followed by attack of water molecule that breaks the phosphovanadate bond to yield dNMP. B, in the active site of Dpo4 two water molecules are observed in close proximity to the primer.