DNA synthesis by a polymerase provides the driving force to accelerate DNA unwinding by a helicase

doi:10.1038/nature03615

Nature. Author manuscript; available in PMC 2006 September 8.

Published in final edited form as:

Nature. 2005 May 19; 435(7040): 370–373.

doi: 10.1038/nature03615

PMCID: PMC1563444

NIHMSID: NIHMS4743

DNA synthesis by a polymerase provides the driving force to accelerate DNA unwinding by a helicase

Natalie M. Stano,¹ Yong-Joo Jeong,^1,² Ilker Donmez,¹ Padmaja Tummalapalli,¹ Mikhail K. Levin,³ and Smita S. Patel¹

¹Department of Biochemistry, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854

²Department of Bio and Nanochemistry, Kookmin University, 861-1, Chongnung-dong, Songbuk-gu, Seoul 136-702, Korea

³Center for Cell Analysis and Modeling, University of Connecticut Health Center, Farmington, CT 06030-1507

Correspondence and requests for materials should be addressed to S.S.P (Email: patelss/at/umdnj.edu)

The publisher's final edited version of this article is available at Nature

See other articles in PMC that cite the published article.

Abstract

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 K_1/2 of 11 μM and V_max 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.

Figure 1

DNA synthesis by T7 DNA polymerase. a, Progressive elongation of the radiolabeled 34-nt primer annealed to the 65-nt template at 100 μM dNTPs. b, The average DNA synthesis rate increases hyperbolically with dNTP concentration (observed rate = (more ...)

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.

Figure 2

T7 helicase’s unwinding rate depends on the speed of DNA synthesis. a, 30-bp replication substrate. b, Unwinding kinetics of the 30-bp region of the replication substrate in the absence (•) or presence of T7 DNA polymerase at 1 mM dTTP (more ...)

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 (K_d ~ 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 K_1/2 identical to the one for DNA synthesis (Fig. 1b) and V_max 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.

Figure 3

DNA synthesis by T7 DNA polymerase and T7 helicase. a, Progressive elongation of the 34-nt primer by T7 DNA polymerase and T7 helicase on the 30-bp replication substrate. b, Kinetics of DNA synthesis by T7 DNA polymerase and the ΔCt T7 helicase (more ...)

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 (dsDNA [right harpoon over left harpoon]

2ssDNA) 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.

Figure 4

DNA synthesis by T4 and T7 DNA polymerases and T7 helicase. Lanes 1–5 in all experiments correspond to reaction times of 0.025, 0.1, 0.5, 1.0, and 3.0 s. a, DNA synthesis by T4 DNA polymerase on the 30-bp replication substrate at 100 μM (more ...)

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.

Methods

DNA

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).

Proteins

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.

DNA unwinding kinetics

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.

Data analysis

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.

(Eq. 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 − L_m) / s, where s is step size, L is the number of base pairs in the DNA substrate duplex, L_m 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

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.

Supplementary Material

Supplementary Figure 1

Click here to view.^{(100K, pdf)}

Acknowledgments

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.

Footnotes

Competing Interests Statement

The authors declare that they have no competing financial interests.

References

1. Patel SS, Picha KM. Structure and function of hexameric helicases. Annu Rev Biochem. 2000;69:651–697. [PubMed]

2. Kim DE, Narayan M, Patel SS. T7 DNA helicase: a molecular motor that processively and unidirectionally translocates along single-stranded DNA. J Mol Biol. 2002;321:807–819. [PubMed]

3. Jeong YJ, Levin MK, Patel SS. The DNA-unwinding mechanism of the ring helicase of bacteriophage T7. Proc Natl Acad Sci USA. 2004;101:7264–9. [PubMed]

4. Richardson CC. Bacteriophage T7: minimal requirements for the replication of a duplex DNA molecule. Cell. 1983;33:315–317. [PubMed]

5. Lee J, Chastain PD, Kusakabe T, Griffith JD, Richardson CC. Coordinated leading and lagging strand DNA synthesis on a minicircular template. Mol Cell. 1998;1:1001–1010. [PubMed]

6. Patel SS, Wong I, Johnson KA. Pre-steady-state kinetic analysis of processive DNA replication including complete characterization of an exonuclease-deficient mutant. Biochemistry. 1991;30:511–25. [PubMed]

7. Matson SW, Richardson CC. DNA-dependent nucleoside 5′-triphosphatase activity of the gene 4 protein of bacteriophage T7. J Biol Chem. 1983;258:14009–14016. [PubMed]

8. Matson SW, Tabor S, Richardson CC. The gene 4 protein of bacteriophage T7. Characterization of helicase activity. J Biol Chem. 1983;258:14017–14024. [PubMed]

9. Patel SS, Rosenberg AH, Studier FW, Johnson KA. Large scale purification and biochemical characterization of T7 primase/helicase proteins. Evidence for homodimer and heterodimer formation. J Biol Chem. 1992;267:15013–15021. [PubMed]

10. Ali JA, Lohman TM. Kinetic measurement of the step size of DNA unwinding by Escherichia coli UvrD helicase. Science. 1997;275:377–380. [PubMed]

11. Notarnicola SM, Mulcahy HL, Lee J, Richardson CC. The acidic carboxyl terminus of the bacteriophage T7 gene 4 helicase/primase interacts with T7 DNA polymerase. J Biol Chem. 1997;272:18425–18433. [PubMed]

12. von Hippel PH, Delagoutte E. A general model for nucleic acid helicases and their “coupling” within macromolecular machines. Cell. 2001;104:177–190. [PubMed]

13. Kim YT, Tabor S, Bortner C, Griffith JD, Richardson CC. Purification and characterization of the bacteriophage T7 gene 2.5 protein. A single-stranded DNA-binding protein. J Biol Chem. 1992;267:15022–31. [PubMed]

14. Lohman TM, Ferrari ME. Escherichia coli single-stranded DNA-binding protein: multiple DNA-binding modes and cooperativities. Annu Rev Biochem. 1994;63:527–70. [PubMed]

15. Delagoutte E, von Hippel PH. Molecular mechanisms of the functional coupling of the helicase (gp41) and polymerase (gp43) of bacteriophage T4 within the DNA replication fork. Biochemistry. 2001;40:4459–77. [PubMed]

16. Levin, M. K. & Patel, S. S. in Molecular motors (ed. Schliwa, M.) 179–198 (Wiley-VCH Verlag GmbH, Weinheim Germany, 2003).

17. Levin MK, Gurjar MM, Patel SS. ATP binding modulates the nucleic acid affinity of hepatitis C virus helicase. J Biol Chem. 2003;278:23311–6. [PubMed]

18. Wang H, Oster G. Ratchets, power strokes, and molecular motors. Applied Physics A. 2002;75:315–323.

19. Kim S, Dallmann HG, McHenry CS, Marians KJ. Coupling of a replicative polymerase and helicase: a tau-DnaB interaction mediates rapid replication fork movement. Cell. 1996;84:643–650. [PubMed]

20. Mok M, Marians KJ. The Escherichia coli preprimosome and DNA B helicase can form replication forks that move at the same rate. J Biol Chem. 1987;262:16644–16654. [PubMed]

21. Korhonen JA, Pham XH, Pellegrini M, Falkenberg M. Reconstitution of a minimal mtDNA replisome in vitro. EMBO J. 2004;23:2423–9. [PubMed]

22. Schrock RD, Alberts B. Processivity of the gene 41 DNA helicase at the bacteriophage T4 DNA replication fork. J Biol Chem. 1996;271:16678–16682. [PubMed]

23. Cha TA, Alberts BM. The bacteriophage T4 DNA replication fork. Only DNA helicase is required for leading strand DNA synthesis by the DNA polymerase holoenzyme. J Biol Chem. 1989;264:12220–5. [PubMed]

24. Lucius AL, Maluf NK, Fischer CJ, Lohman TM. General Methods for Analysis of Sequential “n-step” Kinetic Mechanisms: Application to Single Turnover Kinetics of Helicase-Catalyzed DNA Unwinding. Biophys J. 2003;85:2224–2239. [PubMed]

25. Frey MW, Nossal NG, Capson TL, Benkovic SJ. Construction and characterization of a bacteriophage T4 DNA polymerase deficient in 3′-->5′ exonuclease activity. Proc Natl Acad Sci USA. 1993;90:2579–83. [PubMed]