Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

A Brownian motor mechanism of translocation and strand separation by hepatitis C virus helicase

Nature Structural & Molecular Biology volume 12, pages 429–435 (2005)Cite this article

Abstract

Helicases translocate along their nucleic acid substrates using the energy of ATP hydrolysis and by changing conformations of their nucleic acid–binding sites. Our goal is to characterize the conformational changes of hepatitis C virus (HCV) helicase at different stages of ATPase cycle and to determine how they lead to translocation. We have reported that ATP binding reduces HCV helicase affinity for nucleic acid. Now we identify the stage of the ATPase cycle responsible for translocation and unwinding. We show that a rapid directional movement occurs upon helicase binding to DNA in the absence of ATP, resulting in opening of several base pairs. We propose that HCV helicase translocates as a Brownian motor with a simple two-stroke cycle. The directional movement step is fueled by single-stranded DNA binding energy while ATP binding allows for a brief period of random movement that prepares the helicase for the next cycle.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: DNA substrates.
Figure 2: NS3h affinity to ssDNA and 3′-tail ss/dsDNA substrates.
Figure 3: Stopped-flow kinetics of DNA binding to NS3h.
Figure 4: NS3h-binding rate depends on the length of the ssDNA.
Figure 5: NS3h-binding rate is affected by base pair strength at the ss/dsDNA junction.
Figure 6: Brownian motor mechanism of translocation and unwinding.

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  1. 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  PubMed Central  Google Scholar 

  2. Tai, C.L., Chi, W.K., Chen, D.S. & Hwang, L.H. The helicase activity associated with hepatitis C virus nonstructural protein 3 (NS3). J. Virol. 70, 8477–8484 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Gwack, Y., Kim, D.W., Han, J.H. & Choe, J. DNA helicase activity of the hepatitis C virus nonstructural protein 3. Eur. J. Biochem. 250, 47–54 (1997).

    Article  CAS  Google Scholar 

  4. Frick, D.N., Rypma, R.S., Lam, A.M. & Gu, B. The nonstructural protein 3 protease/helicase requires an intact protease domain to unwind duplex RNA efficiently. J. Biol. Chem. 279, 1269–1280 (2004).

    Article  CAS  Google Scholar 

  5. 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  PubMed Central  Google Scholar 

  6. Howe, A.Y. et al. A novel recombinant single-chain hepatitis C virus NS3-NS4A protein with improved helicase activity. Protein Sci. 8, 1332–1341 (1999).

    Article  CAS  PubMed Central  Google Scholar 

  7. Gallinari, P. et al. Modulation of hepatitis C virus NS3 protease and helicase activities through the interaction with NS4A. Biochemistry 38, 5620–5632 (1999).

    Article  CAS  Google Scholar 

  8. Heilek, G.M. & Peterson, M.G. A point mutation abolishes the helicase but not the nucleoside triphosphatase activity of hepatitis C virus NS3 protein. J. Virol. 71, 6264–6266 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Kim, D.W., Gwack, Y., Han, J.H. & Choe, J. Towards defining a minimal functional domain for NTPase and RNA helicase activities of the hepatitis C virus NS3 protein. Virus Res. 49, 17–25 (1997).

    Article  CAS  Google Scholar 

  10. Kuang, W.F. et al. Hepatitis C virus NS3 RNA helicase activity is modulated by the two domains of NS3 and NS4A. Biochem. Biophys. Res. Commun. 317, 211–217 (2004).

    Article  CAS  Google Scholar 

  11. Levin, M.K., Wang, 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 

  12. Delagoutte, E. & von Hippel, P.H. Helicase mechanisms and the coupling of helicases within macromolecular machines Part I: Structures and properties of isolated helicases. Q. Rev. Biophys. 35, 431–478 (2002).

    Article  CAS  Google Scholar 

  13. von Hippel, P.H. & Delagoutte, E. A general model for nucleic acid helicases and their “coupling” within macromolecular machines. Cell 104, 177–190 (2001).

    Article  CAS  Google Scholar 

  14. Lohman, T.M. & Bjornson, K.P. Mechanisms of helicase-catalyzed DNA unwinding. Annu. Rev. Biochem. 65, 169–214 (1996).

    Article  CAS  Google Scholar 

  15. Patel, S.S. & Picha, K.M. Structure and function of hexameric helicases. Annu. Rev. Biochem. 69, 651–697 (2000).

    Article  CAS  Google Scholar 

  16. Matson, S.W., Bean, D.W. & George, J.W. DNA helicases: enzymes with essential roles in all aspects of DNA metabolism. BioEssays 16, 13–22 (1994).

    Article  CAS  Google Scholar 

  17. Tackett, A.J., Morris, P.D., Dennis, R., Goodwin, T.E. & Raney, K.D. Unwinding of unnatural substrates by a DNA helicase. Biochemistry 40, 543–548 (2001).

    Article  CAS  Google Scholar 

  18. Kawaoka, J., Jankowsky, E. & Pyle, A.M. Backbone tracking by the SF2 helicase NPH-II. Nat. Struct. Mol. Biol. 11, 526–530 (2004).

    Article  CAS  Google Scholar 

  19. Byrd, A.K. & Raney, K.D. Protein displacement by an assembly of helicase molecules aligned along single-stranded DNA. Nat. Struct. Mol. Biol. 11, 531–538 (2004).

    Article  CAS  PubMed Central  Google Scholar 

  20. Morris, P.D. & Raney, K.D. DNA helicases displace streptavidin from biotin-labeled oligonucleotides. Biochemistry 38, 5164–5171 (1999).

    Article  CAS  Google Scholar 

  21. Jankowsky, E., Gross, C.H., Shuman, S. & Pyle, A.M. Active Disruption of an RNA-Protein Interaction by a DExH/D RNA Helicase. Science 291, 121–125 (2001).

    Article  CAS  Google Scholar 

  22. Jeong, Y.J., Levin, M.K. & Patel, S.S. The DNA-unwinding mechanism of the ring helicase of bacteriophage T7. Proc. Natl. Acad. Sci. USA 101, 7264–7269 (2004).

    Article  CAS  Google Scholar 

  23. Hingorani, M.M. & Patel, S.S. Interactions of bacteriophage T7 DNA primase/helicase protein with single-stranded and double-stranded DNAs. Biochemistry 32, 12478–12487 (1993).

    Article  CAS  Google Scholar 

  24. Bujalowski, W. & Jezewska, M.J. Interactions of Escherichia coli primary replicative helicase DnaB protein with single-stranded DNA. The nucleic acid does not wrap around the protein hexamer. Biochemistry 34, 8513–8519 (1995).

    Article  CAS  Google Scholar 

  25. Preugschat, F., Averett, D.R., Clarke, B.E. & Porter, D.J.T. A steady-state and pre-steady-state kinetic analysis of the NTPase activity associated with the hepatitis C virus NS3 helicase domain. J. Biol. Chem. 271, 24449–24457 (1996).

    Article  CAS  Google Scholar 

  26. 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 

  27. Levin, M.K. & Patel, S.S. The helicase from hepatitis C virus is active as an oligomer. J. Biol. Chem. 274, 31839–31846 (1999).

    Article  CAS  Google Scholar 

  28. 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 

  29. Gwack, Y., Kim, D.W., Han, J.H. & Choe, J. Characterization of RNA binding activity and RNA helicase activity of the hepatitis C virus NS3 protein. Biochem. Biophys. Res. Commun. 225, 654–659 (1996).

    Article  CAS  Google Scholar 

  30. Leroy, J.L., Kochoyan, M., Huynh-Dinh, T. & Gueron, M. Characterization of base-pair opening in deoxynucleotide duplexes using catalyzed exchange of the imino proton. J. Mol. Biol. 200, 223–238 (1988).

    Article  CAS  Google Scholar 

  31. Nonin, S., Leroy, J.L. & Gueron, M. Terminal base pairs of oligodeoxynucleotides: imino proton exchange and fraying. Biochemistry 34, 10652–10659 (1995).

    Article  CAS  Google Scholar 

  32. Bonnet, G., Krichevsky, O. & Libchaber, A. Kinetics of conformational fluctuations in DNA hairpin-loops. Proc. Natl. Acad. Sci. USA 95, 8602–8606 (1998).

    Article  CAS  Google Scholar 

  33. 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  CAS  Google Scholar 

  34. Farah, J.A. & Smith, G.R. The RecBCD enzyme initiation complex for DNA unwinding: enzyme positioning and DNA opening. J. Mol. Biol. 272, 699–715 (1997).

    Article  CAS  Google Scholar 

  35. Ali, J.A., Maluf, N.K. & Lohman, T.M. An oligomeric form of E. coli UvrD is required for optimal helicase activity. J. Mol. Biol. 293, 815–834 (1999).

    Article  CAS  Google Scholar 

  36. Nanduri, B., Byrd, A.K., Eoff, R.L., Tackett, A.J. & Raney, K.D. Pre-steady-state DNA unwinding by bacteriophage T4 Dda helicase reveals a monomeric molecular motor. Proc. Natl. Acad. Sci. USA 99, 14722–14727 (2002).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  38. 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 

  39. Astumian, R.D. Thermodynamics and kinetics of a Brownian motor. Science 276, 917–922 (1997).

    Article  CAS  Google Scholar 

  40. Mogilner, A., Wang, H., Elston, T. & Oster, G. Molecular motors: theory and experiment. In Mathematical Biology (eds. Fall, C., Marland, E., Wagner, J. & Tyson, J.) (Springer, New York, 2001).

    Google Scholar 

  41. Wang, H. & Oster, G. Ratchets, power strokes, and molecular motors. Appl. Phys. A 75, 315–323 (2002).

    Article  CAS  Google Scholar 

  42. Ali, J.A. & Lohman, T.M. Kinetic measurement of the step size of DNA unwinding by Escherichia coli UvrD helicase. Science 275, 377–380 (1997).

    Article  CAS  Google Scholar 

  43. Dillingham, M.S., Wigley, D.B. & Webb, M.R. Demonstration of unidirectional single-stranded DNA translocation by PcrA helicase: measurement of step size and translocation speed. Biochemistry 39, 205–212 (2000).

    Article  CAS  Google Scholar 

  44. Dillingham, M.S., Spies, M. & Kowalczykowski, S.C. RecBCD enzyme is a bipolar DNA helicase. Nature 423, 893–897 (2003).

    Article  CAS  Google Scholar 

  45. Kim, D.W., Gwack, Y., Han, J.H. & Choe, J. C-terminal domain of the hepatitis C virus NS3 protein contains an RNA helicase activity. Biochem. Biophys. Res. Commun. 215, 160–166 (1995).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank C.M. Drain, J.-C. Liao, G. Oster and C. Wolgemuth for comments and discussions. This work was supported by US National Institutes of Health grant GM55310 (S.S.P).

Author information

Author notes

  1. Mikhail K Levin

    Present address: Center for Cell Analysis and Modeling, University of Connecticut Health Center, Farmington, Connecticut, 06030-1507, USA

  2. Mikhail K Levin and Madhura Gurjar: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, 08854, New Jersey, USA

    Mikhail K Levin, Madhura Gurjar & Smita S Patel

Authors
  1. Mikhail K Levin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Madhura Gurjar
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Smita S Patel
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Smita S Patel.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Levin, M., Gurjar, M. & Patel, S. A Brownian motor mechanism of translocation and strand separation by hepatitis C virus helicase. Nat Struct Mol Biol 12, 429–435 (2005). https://doi.org/10.1038/nsmb920

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb920

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing