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RNA translocation and unwinding mechanism of HCV NS3 helicase and its coordination by ATP


Helicases are a ubiquitous class of enzymes involved in nearly all aspects of DNA and RNA metabolism. Despite recent progress in understanding their mechanism of action, limited resolution has left inaccessible the detailed mechanisms by which these enzymes couple the rearrangement of nucleic acid structures to the binding and hydrolysis of ATP1,2. Observing individual mechanistic cycles of these motor proteins is central to understanding their cellular functions. Here we follow in real time, at a resolution of two base pairs and 20 ms, the RNA translocation and unwinding cycles of a hepatitis C virus helicase (NS3) monomer. NS3 is a representative superfamily-2 helicase essential for viral replication3, and therefore a potentially important drug target4. We show that the cyclic movement of NS3 is coordinated by ATP in discrete steps of 11 ± 3 base pairs, and that actual unwinding occurs in rapid smaller substeps of 3.6 ± 1.3 base pairs, also triggered by ATP binding, indicating that NS3 might move like an inchworm5,6. This ATP-coupling mechanism is likely to be applicable to other non-hexameric helicases involved in many essential cellular functions. The assay developed here should be useful in investigating a broad range of nucleic acid translocation motors.

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Figure 1: Assay with optical tweezers for assessing the mechanistic cycle of NS3.
Figure 2: [ATP] affects both NS3 pauses and steps.
Figure 3: NS3 steps are composed of substeps.
Figure 4: Effect of force on the behaviour of NS3, and proposed model of action.


  1. Jankowsky, E., Gross, C. H., Shuman, S. & Pyle, A. M. The DExH protein NPH-II is a processive and directional motor for unwinding RNA. Nature 403, 447–451 (2000)

    Article  ADS  CAS  Google Scholar 

  2. Lucius, A. L. & Lohman, T. M. Effects of temperature and ATP on the kinetic mechanism and kinetic step-size for E. coli RecBCD helicase-catalyzed DNA unwinding. J. Mol. Biol. 339, 751–771 (2004)

    Article  CAS  Google Scholar 

  3. Kolykhalov, A. A., Mihalik, K., Feinstone, S. M. & Rice, C. M. Hepatitis C virus-encoded enzymatic activities and conserved RNA elements in the 3′ nontranslated region are essential for virus replication in vivo. J. Virol. 74, 2046–2051 (2000)

    Article  CAS  Google Scholar 

  4. Frick, D. N. Helicases as antiviral drug targets. Drug News Perspect. 16, 355–362 (2003)

    Article  Google Scholar 

  5. Velankar, S. S., Soultanas, P., Dillingham, M. S., Subramanya, H. S. & Wigley, D. B. Crystal structures of complexes of PcrA DNA helicase with a DNA substrate indicate an inchworm mechanism. Cell 97, 75–84 (1999)

    Article  CAS  Google Scholar 

  6. Bianco, P. R. & Kowalczykowski, S. C. Translocation step size and mechanism of the RecBC DNA helicase. Nature 405, 368–372 (2000)

    Article  ADS  CAS  Google Scholar 

  7. Delagoutte, E. & von Hippel, P. H. Helicase mechanisms and the coupling of helicases within macromolecular machines. Part II: Integration of helicases into cellular processes. Q. Rev. Biophys. 36, 1–69 (2003)

    Article  CAS  Google Scholar 

  8. Dimitrova, M., Imbert, I., Kieny, M. P. & Schuster, C. Protein-protein interactions between hepatitis C virus nonstructural proteins. J. Virol. 77, 5401–5414 (2003)

    Article  CAS  Google Scholar 

  9. Pang, P. S., Jankowsky, E., Planet, P. J. & Pyle, A. M. The hepatitis C viral NS3 protein is a processive DNA helicase with cofactor enhanced RNA unwinding. EMBO J. 21, 1168–1176 (2002)

    Article  CAS  Google Scholar 

  10. Levin, M. K., Gurjar, M. & Patel, S. S. A Brownian motor mechanism of translocation and strand separation by hepatitis C virus helicase. Nature Struct. Mol. Biol. 12, 429–435 (2005)

    Article  CAS  Google Scholar 

  11. Kim, J. L. et al. Hepatitis C virus NS3 RNA helicase domain with a bound oligonucleotide: the crystal structure provides insights into the mode of unwinding. Structure 6, 89–100 (1998)

    Article  CAS  Google Scholar 

  12. Dohoney, K. M. & Gelles, J. Chi-sequence recognition and DNA translocation by single RecBCD helicase/nuclease molecules. Nature 409, 370–374 (2001)

    Article  ADS  CAS  Google Scholar 

  13. Bianco, P. R. Processive translocation and DNA unwinding by individual RecBCD enzyme molecules. Nature 409, 374–378 (2001)

    Article  ADS  CAS  Google Scholar 

  14. Ha, T. et al. Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase. Nature 419, 638–641 (2002)

    Article  ADS  CAS  Google Scholar 

  15. Perkins, T. T., Li, H. W., Dalal, R. V., Gelles, J. & Block, S. M. Forward and reverse motion of single RecBCD molecules on DNA. Biophys. J. 86, 1640–1648 (2004)

    Article  ADS  CAS  Google Scholar 

  16. Dessinges, M. N., Lionnet, T., Xi, X. G., Bensimon, D. & Croquette, V. Single-molecule assay reveals strand switching and enhanced processivity of UvrD. Proc. Natl Acad. Sci. USA 101, 6439–6444 (2004)

    Article  ADS  CAS  Google Scholar 

  17. Bustamante, C., Marko, J. F., Siggia, E. D. & Smith, S. Entropic elasticity of lambda-phage DNA. Science 265, 1599–1600 (1994)

    Article  ADS  CAS  Google Scholar 

  18. Levin, M. K., Yang, Y. H. & Patel, S. S. The functional interaction of the hepatitis C virus helicase molecules is responsible for unwinding processivity. J. Biol. Chem. 279, 26005–26012 (2004)

    Article  CAS  Google Scholar 

  19. Singleton, M. R., Dillingham, M. S., Gaudier, M., Kowalczykowski, S. C. & Wigley, D. B. Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks. Nature 432, 187–193 (2004)

    Article  ADS  CAS  Google Scholar 

  20. Serebrov, V. S. & Pyle, A. M. Periodic cycles of RNA unwinding and pausing by hepatitis C virus NS3 helicase. Nature 430, 476–480 (2004)

    Article  ADS  CAS  Google Scholar 

  21. Ali, J. A. & Lohman, T. M. Kinetic measurement of the step size of DNA unwinding by Escherichia coli UvrD helicase. Science 276, 377–380 (1997)

    Article  Google Scholar 

  22. Levin, M. K. & Patel, S. S. Helicase from hepatitis C virus, energetics of DNA binding. J. Biol. Chem. 277, 29377–29385 (2002)

    Article  CAS  Google Scholar 

  23. Levin, M. K., Gurjar, M. M. & Patel, S. S. ATP binding modulates the nucleic acid affinity of hepatitis C virus helicase. J. Biol. Chem. 278, 23311–23316 (2003)

    Article  CAS  Google Scholar 

  24. Tackett, A. J., Chen, Y., Cameron, C. E. & Raney, K. D. Multiple full-length NS3 molecules are required for optimal unwinding of oligonucleotide DNA in vitro. J. Biol. Chem. 280, 10797–10806 (2005)

    Article  CAS  Google Scholar 

  25. Tackett, A. J., Wei, L., Cameron, C. E. & Raney, K. D. Unwinding of nucleic acids by HCV NS3 helicase is sensitive to the structure of the duplex. Nucleic Acids Res. 29, 565–572 (2001)

    Article  CAS  Google Scholar 

  26. Bianco, P. R. Hepatitis C NS3 helicase unwinds RNA in leaps and bounds. Lancet 364, 1385–1387 (2004)

    Article  Google Scholar 

  27. Yao, N. et al. Structure of the hepatitis C virus RNA helicase domain. Nature Struct. Biol. 4, 463–467 (1997)

    Article  CAS  Google Scholar 

  28. Smith, S. B., Cui, Y. & Bustamante, C. Optical-trap force transducer that operates by direct measurement of light momentum. Methods Enzymol. 361, 134–162 (2003)

    Article  CAS  Google Scholar 

  29. Liphardt, J., Onoa, B., Smith, S. B., Tinoco, I. Jr & Bustamante, C. Reversible unfolding of single RNA molecules by mechanical force. Science 292, 733–737 (2001)

    Article  ADS  CAS  Google Scholar 

  30. Bryant, Z. et al. Structural transitions and elasticity from torque measurements on DNA. Nature 424, 338–341 (2003)

    Article  ADS  CAS  Google Scholar 

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We thank H. V. Le from the Schering-Plough Research Institute for the NS3 plasmid; S. B. Smith, P. T. X. Li, Y. R. Chemla and J.-C. Liao for discussions and technical help; T. M. Lohman for critical reading of the manuscript, and members of our laboratories for discussions and critical reading of the manuscript. This research was supported by CIHR and FQRNT doctoral fellowships (S.D.), an NIH postdoctoral fellowship (R.K.B.), NIH (I.T., A.M.P., C.B.), DOE (C.B.), and HHMI grants to investigators A.M.P. and C.B.

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Correspondence to Carlos Bustamante.

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Reprints and permissions information is available at The authors declare no competing financial interests.

Supplementary information

Supplementary Figures

Eight figures showing a variety of NS3 behaviours (Supplementary Figure 1), that the 11 bp step size is intrinsic to NS3 (Supplementary Figures 2 and 3), that two kinetic events are required for NS3 pause exit (Supplementary Figure 4), that NS3 steps consist of rapid 2–5 bp substeps (Supplementary Figure 5), and providing part of the evidence that NS3 acts as a monomer in the present assay (Supplementary Figures 6, 7 and 8). (PDF 904 kb)

Supplementary Discussion

Discussions on the variability in the step size of NS3; the comparison between bulk and single molecule experiments on both theoretical and experimental grounds; the evidence for a single active NS3 monomer; two inchworm model orientations consistent with the data; the role of ATP in coordinating translocator and helix opener movement; thermodynamic considerations on coupling between ATP and duplex unwinding; confirming substrate pairing, geometry and attachment; instrument resolution; and the effects of NS3 binding to the substrate. (PDF 188 kb)

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Dumont, S., Cheng, W., Serebrov, V. et al. RNA translocation and unwinding mechanism of HCV NS3 helicase and its coordination by ATP. Nature 439, 105–108 (2006).

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