Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase (original) (raw)

Nature volume 419, pages 638–641 (2002)Cite this article

Abstract

Helicases are motor proteins that couple conformational changes induced by ATP binding and hydrolysis with unwinding of duplex nucleic acid1,2,3, and are involved in several human diseases. Some function as hexameric rings4, but the functional form of non-hexameric helicases has been debated5,6,7,8,9,10. Here we use a combination of a surface immobilization scheme and single-molecule fluorescence assays—which do not interfere with biological activity—to probe DNA unwinding by the Escherichia coli Rep helicase. Our studies indicate that a Rep monomer uses ATP hydrolysis to move toward the junction between single-stranded and double-stranded DNA but then displays conformational fluctuations that do not lead to DNA unwinding. DNA unwinding initiates only if a functional helicase is formed via additional protein binding. Partial dissociation of the functional complex during unwinding results in interruptions (‘stalls’) that lead either to duplex rewinding upon complete dissociation of the complex, or to re-initiation of unwinding upon re-formation of the functional helicase. These results suggest that the low unwinding processivity observed in vitro for Rep is due to the relative instability of the functional complex. We expect that these techniques will be useful for dynamic studies of other helicases and protein–DNA interactions.

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

Access options

Subscribe to this journal

Receive 51 print issues and online access

$199.00 per year

only $3.90 per issue

Buy this article

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

Additional access options:

Similar content being viewed by others

References

  1. Lohman, T. M. & Bjornson, K. P. Mechanisms of helicase-catalyzed DNA unwinding. Annu. Rev. Biochem. 65, 169–214 (1996)
    Article CAS Google Scholar
  2. Hall, M. C. & Matson, S. W. Helicase motifs: the engine that powers DNA unwinding. Mol. Microbiol. 34, 867–877 (1999)
    Article CAS Google Scholar
  3. von Hippel, P. H. & Delagouette, E. A general model for nucleic acid helicases and their “coupling” within macromolecular machines. Cell 104, 177–190 (2001)
    Article CAS Google Scholar
  4. Patel, S. S. & Picha, K. M. Structure and function of hexameric helicases. Annu. Rev. Biochem. 69, 651–697 (2000)
    Article CAS 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. Mechanic, L. E., Hall, M. C. & Matson, S. W. Escherichia coli DNA helicase II is active as a monomer. J. Biol. Chem. 274, 12488–12498 (1999)
    Article CAS Google Scholar
  7. 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
  8. 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
  9. 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
  10. Cheng, W., Hsieh, J., Brendza, K. M. & Lohman, T. M. E. coli Rep oligomers are required to initiate DNA unwinding in vitro. J. Mol. Biol. 310, 327–350 (2001)
    Article CAS Google Scholar
  11. Bianco, P. R. et al. Processive translocation and DNA unwinding by individual RecBCD enzyme molecules. Nature 409, 374–378 (2001)
    Article ADS CAS Google Scholar
  12. Dohoney, K. M. & Gelles, J. χ-Sequence recognition and DNA translocation by single RecBCD helicase/nuclease molecules. Nature 409, 370–374 (2001)
    Article ADS CAS Google Scholar
  13. Weiss, S. Measuring conformational dynamics of biomolecules by single molecule fluorescence spectroscopy. Nature Struct. Biol. 7, 724–729 (2000)
    Article CAS Google Scholar
  14. Ha, T. et al. Probing the interaction between two single molecules—fluorescence resonance energy transfer between a single donor and a single acceptor. Proc. Natl Acad. Sci. USA 93, 6264–6268 (1996)
    Article ADS CAS Google Scholar
  15. Zhuang, X. W. et al. A single-molecule study of RNA catalysis and folding. Science 288, 2048–2051 (2000)
    Article ADS CAS Google Scholar
  16. Ha, T. Single molecule fluorescence resonance energy transfer. Methods 25, 78–86 (2001)
    Article CAS Google Scholar
  17. Sofia, S. J., Premnath, V. & Merrill, E. W. Poly(ethylene oxide) grafted to silicon surfaces: grafting density and protein adsorption. Macromolecules 31, 5059–5070 (1998)
    Article ADS CAS Google Scholar
  18. Chao, K. & Lohman, T. M. DNA-induced dimerization of the Escherichia coli Rep helicase. J. Mol. Biol. 221, 1165–1181 (1991)
    Article CAS Google Scholar
  19. Bjornson, K. P., Amaratunga, M., Moore, K. J. M. & Lohman, T. M. Single-turnover kinetics of helicase-catalyzed DNA unwinding monitored continuously by fluorescence energy transfer. Biochemistry 33, 14306–14316 (1994)
    Article CAS Google Scholar
  20. Moore, K. J. M. & Lohman, T. M. Kinetic mechanism of adenine nucleotide binding to and hydrolysis by the Escherichia coli Rep monomer. 1. Use of fluorescent nucleotide analogues. Biochemistry 33, 14550–14564 (1994)
    Article CAS Google Scholar
  21. Wong, I. & Lohman, T. M. A two-site mechanism for ATP hydrolysis by the asymmetric Rep dimer P2S as revealed by site-specific inhibition with ADP-AlF4. Biochemistry 36, 3115–3125 (1997)
    Article CAS Google Scholar
  22. Dillingham, M. S., Wigley, D. B. & Webb, M. R. Direct measurement of single-stranded DNA translocation by PcrA helicase using the fluorescent base analogue 2-aminopurine. Biochemistry 41, 643–651 (2002)
    Article CAS Google Scholar
  23. Yarranton, G. T. & Gefter, M. L. Enzyme-catalyzed DNA unwinding: studies on Escherichia coli rep protein. Proc. Natl Acad. Sci. USA 76, 1658–1662 (1979)
    Article ADS CAS Google Scholar
  24. Lohman, T. M., Chao, K., Green, J. M., Sage, S. & Runyon, G. T. Large-scale purification and characterization of the Escherichia coli rep gene product. J. Biol. Chem. 264, 10139–10147 (1989)
    CAS PubMed Google Scholar

Download references

Acknowledgements

We thank C. Chidsey for suggesting the use of a PEG surface, and T. Ho for synthesis and purification of the oligonucleotides. This work was supported by the NSF and AFOSR (S.C.), by the NIH (T.M.L.), and by a Searle scholars award, the NIH, an NSF CAREER award, the Research Corporation, and the UIUC research board (T.H.)

Author information

Authors and Affiliations

  1. Department of Physics, University of Illinois, Urbana, Illinois, 61801, USA
    Taekjip Ha & Ivan Rasnik
  2. Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, Missouri, 63110, USA
    Wei Cheng, George H. Gauss & Timothy M. Lohman
  3. Department of Physics, Stanford University, Stanford, California, 94305, USA
    Hazen P. Babcock & Steven Chu

Authors

  1. Taekjip Ha
    You can also search for this author inPubMed Google Scholar
  2. Ivan Rasnik
    You can also search for this author inPubMed Google Scholar
  3. Wei Cheng
    You can also search for this author inPubMed Google Scholar
  4. Hazen P. Babcock
    You can also search for this author inPubMed Google Scholar
  5. George H. Gauss
    You can also search for this author inPubMed Google Scholar
  6. Timothy M. Lohman
    You can also search for this author inPubMed Google Scholar
  7. Steven Chu
    You can also search for this author inPubMed Google Scholar

Corresponding author

Correspondence toTaekjip Ha.

Ethics declarations

Competing interests

The authors declare that they have no competing financial interests.

Supplementary information

Rights and permissions

About this article

Cite this article

Ha, T., Rasnik, I., Cheng, W. et al. Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase.Nature 419, 638–641 (2002). https://doi.org/10.1038/nature01083

Download citation

This article is cited by