Advertisement

Helicases pp 85-98 | Cite as

Protein Displacement by Helicases

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 587)

Abstract

Helicases are ubiquitous enzymes that are vital to all living organisms. They are motor proteins that move in a specific direction along the nucleic acid and unwind the nucleic acid (DNA and RNA). ATP hydrolysis provides energy for helicase translocation and unwinding. The unwinding process provides ssDNA intermediates necessary for replication, recombination, and repair. Mutations in specific DNA helicases can lead to disruption in DNA metabolism. For example, mutations in helicases genes resulted in diseases such as xeroderma pigmentosum, cockayne’s syndrome, Bloom’s syndrome, and Werner’s syndrome. During unwinding, helicases are most likely to encounter proteins while moving along the nucleic acid. Several different research groups have demonstrated that helicases shift or displace proteins from one nucleic acid-bound location to another. These protein–protein collisions could result in displacement of proteins from nucleic acid or dissociation of helicase from nucleic acid. This report describes several different methods developed to study protein displacement by DNA and RNA helicases.

Key words

DNA helicase RNA helicase FRET 
This is a preview of subscription content, log in to check access.

References

  1. 1.
    Delagoutte E. and von Hippel P. H. (2002) Helicase mechanisms and the coupling of helicases within macromolecular machines. Part I: Structures and properties of isolated helicases. Q. Rev. Biophys. 35, 431–478.PubMedCrossRefGoogle Scholar
  2. 2.
    Lohman T. M. and Bjornson K. P. (1996) Mechanisms of helicase-catalyzed DNA unwinding. Annu. Rev. Biochem. 65, 169–214.PubMedCrossRefGoogle Scholar
  3. 3.
    Soultanas P. and Wigley D. B. (2001) Unwinding the ‘Gordian knot’ of helicase action. Trends Biochem. Sci. 26, 47–54.PubMedCrossRefGoogle Scholar
  4. 4.
    Matson S. W., Bean D. W., and George J. W. (1994) DNA helicases: enzymes with essential roles in all aspects of DNA metabolism. BioEssays 16, 13–22.PubMedCrossRefGoogle Scholar
  5. 5.
    Lahue E. E. and Matson S. W. (1988). Escherichia coli DNA helicase I catalyzes a uniderctional and highly processive unwinding reaction. J. Biol. Chem. 263, 3208–3215.PubMedGoogle Scholar
  6. 6.
    Delagoutte E. and von Hippel P. H. (2002) Helicase mechanisms and the coupling of helicases within macromolecular machines. Part I:structures and properties of isolated helicases. Q. Rev. Biophys. 35, 431–478.PubMedCrossRefGoogle Scholar
  7. 7.
    Eggleston A. K., O’Neill T. E., Bradbury E. M., and Kowalczykowski S. C. (1995) Unwinding of nucleosomal DNA by a DNA helicase. J. Biol. Chem. 270, 2024–2031.PubMedCrossRefGoogle Scholar
  8. 8.
    Morris P. D. and Raney K. D. (1999) DNA helicases displace streptavidin from biotin-labeled oligonucleotides. Biochemistry 38, 5164–5171.PubMedCrossRefGoogle Scholar
  9. 9.
    Morris P. D., Byrd A. K., Tackett A. J., Cameron C. E., Tanega P., Ott R., et al. (2002) Hepatitis C virus NS3 and simian virus 40 T antigen helicases displace streptavidin from 5′-biotinylated oligonucleotides but not from 3′-biotinylated oligonucleotides: evidence for directional bias in translocation on single-stranded DNA. Biochemistry 41, 2372–2378.PubMedCrossRefGoogle Scholar
  10. 10.
    Byrd A. K. and Raney K. D. (2004) Protein displacement by an assembly of helicase molecules aligned along single-stranded DNA. Nat. Struct. Mol. Biol. 11, 531–538.PubMedCrossRefGoogle Scholar
  11. 11.
    Byrd A. K. and Raney K. D. (2006) Displacement and unwinding of trp repressor by Dda helicase. Nucleic Acids Res. 34, 3020–3029.PubMedCrossRefGoogle Scholar
  12. 12.
    Anand S. P., Zheng H., Bianco P. R., Leuba S. H., and Khan S. A. (2007) DNA helicase activity of PcrA is not required for the displacement of RecA protein from DNA or inhibition of RecA-mediated strand exchange. J. Bact. 189, 4502–4509.PubMedCrossRefGoogle Scholar
  13. 13.
    Fairman M. E., Maroney P. A., Wang W., Bowers H. A., Gollnick P., Nilsen T. W., et al. (2004) Protein displacement by DExH/D “RNA helicases” with out duplex unwinding. Science 304, 730–734.PubMedCrossRefGoogle Scholar
  14. 14.
    Flores M. J., Sanchez N., and Michel B. (2005) A fork-clearing role for UvrD. Mol. Microbiol. 57, 1664–1675.PubMedCrossRefGoogle Scholar
  15. 15.
    Macris M. A. and Sung P. (2005) Multifaceted role of the Saccharomyces cerevisiae Srs2 helicase in homologous recombination regulation. Biochem. Soc. Trans. 33, 1447–1450.PubMedCrossRefGoogle Scholar
  16. 16.
    Jankowsky E., Gross C. H., Shuman S., and Pyle A. M. (2001) Active disruption of an RNA-protein interaction by a DExH/D RNA helicase. Science 291, 121–125.PubMedCrossRefGoogle Scholar
  17. 17.
    Fyodorov D. V. and Kadonaga J. T. (2001) The many faces of chromatin remodeling: switching beyond transcription. Cell 106, 523–525.PubMedCrossRefGoogle Scholar
  18. 18.
    Lusser A. and Kadonaga J. T. (2003) Chromatin remodeling by ATP-dependent molecular machines. Bioessays 25, 1192–1200.PubMedCrossRefGoogle Scholar
  19. 19.
    Sprouse O. R., Brenowitz M., and Auble D. T. (2006) Snf2/Swi2-related ATPase Mot1 drives displacement of TATA-binding protein by gripping DNA. EMBO J. 25, 1492–1504.PubMedCrossRefGoogle Scholar
  20. 20.
    Park J., Marr M. T., and Roberts J. W. (2002) Escherichia coli transcription repair coupling factor (Mfd protein) rescues arrested complexes by promoting forward translocation. Cell 109, 757–767.PubMedCrossRefGoogle Scholar
  21. 21.
    Gohara D. W., Ha C. S., Kumar S., Gosh B., Arnold J. J., Wisniewki T. J., et al. (1999) Production of “authentic” poliovirus RNA-dependent RNA polymerase (3D(pol)) by ubiquitin-protease-mediated cleavage in Escherichia coli. Protein. Expr. Purif. 17,128–138.PubMedCrossRefGoogle Scholar
  22. 22.
    Tackett A. J., Wei L., Cameron C. E., and Raney K. D. (2001) Unwinding of nucleic acids by HCV NS3 helicase is sensitive to the structure of the duplex. Nucleic Acids Res. 29, 565–572.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular BiologyUniversity of Arkansas for Medical SciencesLittle RockUSA

Personalised recommendations

Cite protocol

Buy options