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J. Biol. Chem., Vol. 283, Issue 17, 11516-11525, April 25, 2008
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From the
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, the Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas, and the ¶Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802
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 DNA unwinding 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 full-length NS3 is part of an oligomeric species in vitro.
Received for publication, October 1, 2007 , and in revised form, January 31, 2008.
* This work was supported by National Institutes of Health (NIH) Grant AI060563 (to K. D. R., J. S., and C. E. C.) and NIH Centers of Biomedical Research Excellence Grant P20 RR15569 (to F. Millet). Core facility support was provided by Arkansas IDeA Network of Biomedical Research Excellence Research Grant P20RR016460 and by the Arkansas Biosciences Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1 and Figs. 1–4.
1 Both authors contributed equally to this work.
2 Present address: Amgen, Inc., One Amgen Center Dr., Mail Stop 14-2-A, Thousand Oaks, CA 91320.
3 To whom correspondence should be addressed. Tel.: 501-686-5244; Fax: 501-686-8169; E-mail: raneykevind{at}uams.edu.
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