Hepatitis C virus NS3 helicase forms oligomeric structures that exhibit optimal DNA unwinding activity in vitro

Bartek Sikora, Yingfeng Chen, Cheryl F. Lichti, Melody K. Harrison, Thomas A. Jennings, Yong Tang, Alan J. Tackett, John B. Jordan, Joshua Sakon, Craig Eugene Cameron, Kevin D. Raney

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

HCV NS3 helicase exhibits activity toward DNA and RNA substrates. The DNA helicase activity of NS3 has been proposed to be optimal when multiple NS3 molecules are bound to the same substrate molecule. NS3 catalyzes little or no measurable DNAunwinding under single cycle conditions in which the concentration of substrate exceeds the concentration of enzyme by 5-fold. However, when NS3 (100 nM) is equimolar with the substrate, a small burst amplitude of ∼8 nM is observed. The burst amplitude increases as the enzyme concentration increases, consistent with the idea that multiple molecules are needed for optimal unwinding. Protein-protein interactions may facilitate optimal activity, so the oligomeric properties of the enzyme were investigated. Chemical cross-linking indicates that full-length NS3 forms higher order oligomers much more readily than the NS3 helicase domain. Dynamic light scattering indicates that full-length NS3 exists as an oligomer, whereas NS3 helicase domain exists in a monomeric form in solution. Size exclusion chromatography also indicates that full-length NS3 behaves as an oligomer in solution, whereas the NS3 helicase domain behaves as a monomer. When NS3 was passed through a small pore filter capable of removing protein aggregates, greater than 95% of the protein and the DNA unwinding activity was removed from solution. In contrast, only ∼10% of NS3 helicase domain and ∼20% of the associated DNA unwinding activity was removed from solution after passage through the small pore filter. The results indicate that the optimally active form of fulllength NS3 is part of an oligomeric species in vitro.

Original languageEnglish (US)
Pages (from-to)11516-11525
Number of pages10
JournalJournal of Biological Chemistry
Volume283
Issue number17
DOIs
StatePublished - Apr 25 2008

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Viruses
Hepacivirus
Oligomers
DNA Helicases
DNA
Substrates
Molecules
Enzymes
Size exclusion chromatography
Dynamic light scattering
Gel Chromatography
Proteins
Monomers
RNA
In Vitro Techniques

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Sikora, B., Chen, Y., Lichti, C. F., Harrison, M. K., Jennings, T. A., Tang, Y., ... Raney, K. D. (2008). Hepatitis C virus NS3 helicase forms oligomeric structures that exhibit optimal DNA unwinding activity in vitro. Journal of Biological Chemistry, 283(17), 11516-11525. https://doi.org/10.1074/jbc.M708125200
Sikora, Bartek ; Chen, Yingfeng ; Lichti, Cheryl F. ; Harrison, Melody K. ; Jennings, Thomas A. ; Tang, Yong ; Tackett, Alan J. ; Jordan, John B. ; Sakon, Joshua ; Cameron, Craig Eugene ; Raney, Kevin D. / Hepatitis C virus NS3 helicase forms oligomeric structures that exhibit optimal DNA unwinding activity in vitro. In: Journal of Biological Chemistry. 2008 ; Vol. 283, No. 17. pp. 11516-11525.
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Sikora, B, Chen, Y, Lichti, CF, Harrison, MK, Jennings, TA, Tang, Y, Tackett, AJ, Jordan, JB, Sakon, J, Cameron, CE & Raney, KD 2008, 'Hepatitis C virus NS3 helicase forms oligomeric structures that exhibit optimal DNA unwinding activity in vitro', Journal of Biological Chemistry, vol. 283, no. 17, pp. 11516-11525. https://doi.org/10.1074/jbc.M708125200

Hepatitis C virus NS3 helicase forms oligomeric structures that exhibit optimal DNA unwinding activity in vitro. / Sikora, Bartek; Chen, Yingfeng; Lichti, Cheryl F.; Harrison, Melody K.; Jennings, Thomas A.; Tang, Yong; Tackett, Alan J.; Jordan, John B.; Sakon, Joshua; Cameron, Craig Eugene; Raney, Kevin D.

In: Journal of Biological Chemistry, Vol. 283, No. 17, 25.04.2008, p. 11516-11525.

Research output: Contribution to journalArticle

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AU - Sikora, Bartek

AU - Chen, Yingfeng

AU - Lichti, Cheryl F.

AU - Harrison, Melody K.

AU - Jennings, Thomas A.

AU - Tang, Yong

AU - Tackett, Alan J.

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AU - Sakon, Joshua

AU - Cameron, Craig Eugene

AU - Raney, Kevin D.

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N2 - HCV NS3 helicase exhibits activity toward DNA and RNA substrates. The DNA helicase activity of NS3 has been proposed to be optimal when multiple NS3 molecules are bound to the same substrate molecule. NS3 catalyzes little or no measurable DNAunwinding under single cycle conditions in which the concentration of substrate exceeds the concentration of enzyme by 5-fold. However, when NS3 (100 nM) is equimolar with the substrate, a small burst amplitude of ∼8 nM is observed. The burst amplitude increases as the enzyme concentration increases, consistent with the idea that multiple molecules are needed for optimal unwinding. Protein-protein interactions may facilitate optimal activity, so the oligomeric properties of the enzyme were investigated. Chemical cross-linking indicates that full-length NS3 forms higher order oligomers much more readily than the NS3 helicase domain. Dynamic light scattering indicates that full-length NS3 exists as an oligomer, whereas NS3 helicase domain exists in a monomeric form in solution. Size exclusion chromatography also indicates that full-length NS3 behaves as an oligomer in solution, whereas the NS3 helicase domain behaves as a monomer. When NS3 was passed through a small pore filter capable of removing protein aggregates, greater than 95% of the protein and the DNA unwinding activity was removed from solution. In contrast, only ∼10% of NS3 helicase domain and ∼20% of the associated DNA unwinding activity was removed from solution after passage through the small pore filter. The results indicate that the optimally active form of fulllength NS3 is part of an oligomeric species in vitro.

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