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Helicases are molecular motors that use the energy of NTP hydrolysis to translocate along a nucleic acid strand and catalyze reactions such as DNA unwinding. The ring-shaped helicase1 of bacteriophage T7 translocates along single stranded (ss) DNA at a speed of 130 base per second2. However, T7 helicase slows down nearly 10-fold when unwinding the strands of duplex DNA3. Here we report that T7 DNA polymerase, unable to catalyze strand displacement DNA synthesis by itself, can increase the unwinding rate to 114 base pairs per second, bringing the helicase to similar speeds as along ssDNA. The helicase-rate stimulation depends upon the DNA synthesis rate and does not rely on specific interactions between the helicase and the polymerase. Efficient duplex DNA synthesis is achieved only by the combined action of the helicase and polymerase. The DNA polymerase depends on the unwinding activity of the helicase that provides ssDNA template. The rapid trapping of the ssDNA bases by the DNA synthesis activity of the polymerase in turn drives the helicase to move forward through duplex DNA at speeds similar to those observed along ssDNA.
The DNA factory of bacteriophage T7 is one of the simplest and widely used as a model system for studying replication mechnisms4. The T7 replication complex, containing a helicase (T7 gp4), a DNA polymerase (T7 gp5 complexed with E. coli thioredoxin), and a ssDNA binding protein (T7 gp2.5), efficiently catalyses leading and lagging strand DNA synthesis5. The polymerase alone can elongate a DNA primer when the downstream DNA template is single-stranded (Fig. 1a). The average rate of DNA synthesis by T7 DNA polymerase increases in a hyperbolic manner with dNTP concentration with a K1/2 of 11 μM and Vmax of 230 nt s−1 (nucleotide per second) at 18 °C (Fig. 1b), which is consistent with previous pre-steady state kinetic measurements6. DNA synthesis is blocked when the downstream template DNA is duplex (Fig. 1c). T7 DNA polymerase incorporates only 4 to 5 nt on the duplex template before DNA synthesis stalls. These results indicate that T7 DNA polymerase cannot unwind the duplex DNA beyond 4 to 5 bp and hence cannot catalyse strand displacement DNA synthesis.
T7 helicase uses the energy of dTTP hydrolysis for translocation and unwinding of duplex DNA3,7–9. Using an all-or-none radiometric assay carried out under single-turnover conditions10, we measured the unwinding activity of T7 helicase on the 30-bp replication substrate (Fig. 2a,b). T7 helicase was preincubated with the replication substrate (Fig. 2a) in the presence of dTTP without Mg2+ (conditions that allow assembly of the protein on the DNA, but no unwinding), and reaction was started by rapid addition of Mg2+. T7 helicase unwinds the replication substrate at an average rate of 9 bp s−1 in the absence of T7 DNA polymerase (Fig. 2b). In the presence of T7 DNA polymerase, saturating dTTP, with the rest of the 3 dNTPs at 100 μM, conditions under which T7 DNA polymerase is capable of incorporating nucleotides at its maximal rate, the unwinding rate of T7 helicase was 114 bp s−1 (Fig, 2b,c). This is about a 13-fold enhancement in the unwinding rate of T7 helicase.
We find that the helicase rate stimulation depends on the rate of DNA synthesis, which, in turn depends on dNTP concentrations (Fig 1b). T7 helicase uses only dTTP as an energy source for unwinding (Kd ~ 80 μM)3 and other dNTPs do not support DNA unwinding8. To investigate the effect of DNA synthesis rate on helicase rate stimulation, we varied the concentration of 3 dNTPs while keeping dTTP at 1 mM. The observed unwinding rate increases with increasing dNTP in a hyperbolic fashion with a K1/2 identical to the one for DNA synthesis (Fig. 1b) and Vmax of 130 bp s−1(Fig. 2c). These results show that a) the unwinding rate of T7 helicase depends on the speed of T7 DNA polymerase, and b) when the DNA polymerase can incorporate nucleotides at a rate much faster than the translocation rate of the helicase, it stimulates the helicase to unwind DNA at the rate of its translocation along ssDNA (130 nt s−1)2.
The coupled action of the helicase and polymerase can be monitored by the primer elongation assay. We show that the 30-bp DNA substrate is not replicated efficiently by T7 DNA polymerase alone (Fig. 1c). T7 DNA polymerase, however, replicates this substrate efficiently in the presence of T7 helicase (Fig. 3a). The time course of base additions shows that about 18 nt are added in 150 ms. This corresponds to a maximum rate of DNA synthesis of about 120 nt s−1 by the helicase-polymerase complex. This rate is comparable to the one measured by the all-or-none unwinding assay (Fig. 2b,c). Thus, both the DNA unwinding assay and the DNA synthesis assay show that T7 DNA polymerase activates T7 helicase.
The activities of T7 helicase and T7 DNA polymerase are interdependent. The DNA polymerase requires the helicase to provide it with the ssDNA template for DNA synthesis. Similarly, the DNA polymerase serves to increase the unwinding rate of the helicase. In addition, the experiments show that the rate of DNA synthesis dictates the stimulated unwinding rate. What is the mechanism of this stimulation? Since T7 helicase and T7 DNA polymerase specifically interact to form a complex, one possibility is that the rate increase occurs through an allosteric change in the helicase producing a better unwinding motor.
T7 helicase forms a specific complex with T7 DNA polymerase through 17 C-terminal amino acid residues 11. To test the role of the specific complex formation, we prepared a ΔCt mutant of T7 helicase that lacks the 17 residues. The ΔCt mutant of T7 helicase has both wild-type ssDNA-stimulated dTTPase and helicase activities11, translocates along ssDNA with a rate similar to the wild type helicase (data not shown), but was shown not to form a stable complex with the polymerase11. Rapid DNA synthesis was observed in the presence of ΔCt T7 helicase mutant (Fig. 3b). Based on the addition of 16 nt in 100 msec, we estimate that the 30-bp duplex is replicated with a rate of about 160 nt s−1. Therefore the interactions between T7 DNA polymerase and the C-terminal residues of T7 helicase are not the crucial determinant of the stimulation for the helicase reaction. Note that the interaction between T7 helicase and T7 DNA polymerase serve to increase the processivity of the complex that becomes critical for copying long DNA templates11. T7 helicase and T7 DNA polymerase possess a sufficient degree of intrinsic processivity to unwind and replicate the short DNA duplexes used here in the absence of specific interactions. A smaller fraction of DNA primer was elongated initially with the ΔCt T7 helicase indicating that specific interactions also play a role in the initial complex assembly.
Any protein that binds tightly to ssDNA can shift the equilibrium of the DNA unwinding reaction (dsDNA2ssDNA) towards ssDNA12. Therefore, the DNA polymerase could facilitate the helicase unwinding activity by trapping the complementary strand that is displaced by the helicase. Consequently, we investigated if ssDNA binding proteins would have the same effect. We used a fork DNA as an unwinding substrate that contained a T15 3′ -ssDNA tail, and T7 gp2.513 and E. coli SSB14 proteins that bind ssDNA with a high affinity. The results (supplementary Fig. 1) show that the ssDNA binding proteins have little effect on the unwinding rate. A modest increase in the initial unwinding amplitude was observed, which suggests that the processivity of unwinding is higher in the presence of the ssDNA binding proteins.
The experiments with ssDNA-binding proteins indicate that helicase stimulation by T7 DNA polymerase must involve more than simply binding of the protein to the displaced complementary strand. Although both DNA polymerase and SSB proteins capture the nascent DNA strand, the polymerase does so more rapidly and with an increment of just one base. The fact that the T7 helicase stimulation depends on the speed of the DNA polymerase substantiates this hypothesis. Our model predicts that a heterologous DNA polymerase that is as fast and processive as T7 DNA polymerase should also be able to stimulate the unwinding rate of T7 helicase. This was in fact observed in a study where T7 DNA polymerase was shown to support efficient duplex DNA replication with the T4 helicase15.
To test the ability of a heterologous polymerase to stimulate unwinding, we used T4 gp43 core DNA polymerase, which does not have strand displacement activity (Fig. 4a). In the presence of T7 helicase, T4 DNA polymerase catalyses strand displacement DNA synthesis at a rate of about 30 nt s−1 (Fig. 4b). Thus, T4 DNA polymerase provides a 3-fold rate increase. The lower degree of stimulation is consistent with the slower speed of T4 DNA polymerase on a ssDNA template (~50 nt s−1) (Fig. 4c). To compensate for the different intrinsic synthesis rates, the optimal stimulated rate of the T4 polymerase-helicase should be compared with the rate of T7 polymerase-helicase measured at 1.8 μM (~50–60 nt s−1, compare Fig. 4e and 4c), the conditions providing the same intrinsic synthesis rate. Under these conditions, T7 DNA polymerase stimulates T7 helicase to the same extent as T4 DNA polymerase (compare Fig. 4d and 4b). The fraction of DNA primer elongated to full-length product is less with the heterologous T4 DNA polymerase, consistent with the idea that specific interactions are important for initial complex assembly. These results indicate that a heterologous DNA polymerase and helicase can show cooperativity in DNA synthesis and unwinding, and that the extent of the helicase activity stimulation depends upon the intrinsic rate of DNA synthesis.
The reduced translocation rate of the helicase during unwinding and its stimulation by polymerases can be explained in terms of a Brownian motor mechanism16–18. The Brownian motor, helicase, translocates along the DNA strand through a succession of power strokes and diffusion states. While in the diffusion state the helicase can move in either direction along the DNA, the power strokes make the net movement unidirectional. In the presence of the complimentary strand, DNA base pairs fluctuating between closed and open states produce a net force directed against the helicase forward motion. The power strokes of the helicase can overcome the force, however, during the diffusion phase, backward motion occurs frequently. Therefore, the observed DNA unwinding rate is slower than the helicase’s intrinsic translocation rate along ssDNA3.
T7 and T4 DNA polymerases lack strand displacement activity (unwinding) beyond 4–5 bp. Thus, the activities of these DNA polymerases are dependent on the unwinding activity of the helicase that provides a ssDNA template. The action of the DNA polymerase, that is the formation of new duplex DNA, in turn results in the efficient trapping of the DNA strand created by the helicase. The forward steps of the DNA polymerase may serve to increase the helicase’s net forward movement (“push”) or to decrease the helicase’s backward slips (“brake”). The observed unwinding rate depends on the speed of DNA synthesis. Only when the polymerase rapidly traps the ssDNA bases as soon as they are formed by the helicase, can the helicase obtain its maximal rate, and the complex is able to move through duplex at speeds similar to those observed for the helicase along ssDNA.
The synergy between the helicase and polymerase is important in DNA replication and is also evident from studies of replication complexes of E. coli19,20, human mitochondria21 and phage T415,22,23. The studies of the simpler T7 system continues to provide a deeper understanding of the enzymatic mechanisms of DNA replication many of which are likely to be generally applicable to the more complex replication systems.
Oligodeoxynucleotides (Integrated DNA Technologies, Coralville, IA) were purified by PAGE (polyacrylamide gel electrophoresis), and DNA concentrations were determined in 8M urea (260 nm absorbance and calculated extinction coefficients). DNA was radiolabeled at the 5′-end using γ(32P)ATP and T4 polynucleotide kinase. The replication substrate was made by annealing the 5′-strand (5′ -T35-GAG CGG ATT ACT ATA CTA CAT TAG AAT TCA), 34-nt primer (5′ -CTA GTT ACA GAG TTA TGG TGA CGA TAC AAA CTA T), and the 3′-strand (3′ -GAT CAA TGT CTC AAT ACC ACT GCT ATG TTT GAT ATC TCG CCT AAT GAT ATG ATG TAA TCT TAA GT). The fork DNA was made by annealing the 5′ -strand with the T15-3′ -strand (3′ -T15-CTC GCC TAA TGA TAT GAT GTA ATC TTA AGT).
T7 helicase (gp4A′), a M64L mutant of T7 helicase-primase protein, was purified as described previously9. T7 gp5 (D5A, E7A exo− gp5) was purified as described6 and E. coli thioredoxin was purchased from Sigma Chemicals. The ΔCt T7 helicase was prepared as described11. T7 gp2.5 was purified as described13, E. coli SSB was a kind gift from Mike O’Donnell (Rockefeller U.), and T4 DNA polymerase exo- mutant (D219A)25 was a kind gift from Anthony Berdis (Case Western Reserve Medical School). Protein concentrations were determined by absorbance measurements at 280 nm in 8 M urea using their calculated extinction coefficients.
Unwinding kinetics was measured in a RQF3 rapid quench-flow instrument (KinTek) at 18 °C3. T7 helicase, DNA substrate (with radiolabeled 5′ -strand), 2 mM dTTP, and 3 mM EDTA in buffer T (50 mM Tris-Cl, pH 7.5, 40 mM NaCl, 10 % glycerol) was loaded in one syringe of the quench-flow apparatus. MgCl2 (final free concentration of 4 mM), dNTPs (various concentrations), and trap (3 μM of unlabeled 5′-strand) was added from the second syringe to initiate the reaction. The reactions were quenched after predetermined times, resolved by native PAGE, and products analyzed as described previously3. The unwinding reactions contained a final 200 nM T7 helicase hexamer and 2.5 nM of 30-bp replication substrate. The unwinding reactions with 200 nM T7 DNA polymerase contained 2 μM E. coli thioredoxin, 200 nM T7 helicase, and 100 nM replication substrate. Unwinding reactions with ssDNA binding protein contained 5 μM T7 gp2.5 or 1 μM of E. coli SSB and these were added with MgCl2 at the start of the reaction. No DNA trap was added with the MgCl2 when ssDNA binding protein was present, but the trap was added with the quenching solution to prevent the unwound strands from reannealing.
Kinetic fitting was performed using MATLAB with Optimization toolbox software (The MathWorks, Inc., Natick, MA). Stepwise unwinding kinetics was described using the incomplete gamma function 3,10,24. Best fits were obtained assuming unwinding by two populations of helicase species with identical step size s, but different stepping rates (k1 and k2). The stepping kinetics was described by Equation 1.
Where F is fraction of unwound DNA substrate molecules, A1 and A2 are the amplitudes of unwinding, incomplete gamma function, Γ(k, t, n) = 1/integral(from 0 to Inf)(exp(−x)x^(n−1)dx) * integral(from 0 to kt)(exp(−x)x^(n−1)dx), and t is reaction time. The number of steps, n = (L − Lm) / s, where s is step size, L is the number of base pairs in the DNA substrate duplex, Lm is the length of the shortest DNA duplex that can stay together under the experimental conditions and it was fixed to 12-bp, based on previous studies3.
DNA synthesis kinetics were measured using the rapid quench-flow instrument at 18 °C. The DNA substrate with a radiolabeled 34-nt primer (100 nM) was incubated with the DNA polymerase (200 nM) and E. coli thioredoxin (2 μM with T7 DNA polymerase) with or without T7 helicase or ΔCt T7 helicase (200 nM hexamer) in buffer T containing dTTP (1 mM) and EDTA (1.5 mM). This solution was mixed rapidly with dNTPs (various concentrations) and MgCl2 (free final Mg2+ concentration was 4 mM) in buffer T to initiate the reaction. After predetermined times, reactions were quenched (200 mM EDTA), resolved by electrophoresis on a 14 or 16 % polyacrylamide/7M urea gel (0.25–0.75 mm wedge spacers), and visualized and quantitated using the PhosphorImager (ImageQuant software). The average rate of DNA synthesis was determined from the exponential increase in DNA products with time ([DNA products] = A × (1−e−kt), where A is the amplitude, t is time, and k is the observed DNA synthesis rate.
We thank Mike O’Donnell, Anthony Berdis, Nathalie Andraos, Charles C. Richardson, and Margarita Salas for the kind gift of proteins, and Charles M. Drain for critical reading of the manuscript. This research was supported by NIH grant GM55310 to SSP.
Competing Interests Statement
The authors declare that they have no competing financial interests.