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J Biol Chem. 2009 Jan 23;284(4):2512-21. doi: 10.1074/jbc.M805460200. Epub 2008 Nov 14.

Establishing a mechanistic basis for the large kinetic steps of the NS3 helicase.

Author information

1
Department of Molecular Biophysics and Biochemistry and Howard Hughes Medical Institute, Yale University, New Haven, Connecticut 06520, USA.

Abstract

The NS3 helicase from hepatitis C virus is a prototypical DEx(H/D) RNA helicase. NS3 has been shown to unwind RNA in a discontinuous manner, pausing after long apparent steps of unwinding. We systematically examined the effects of duplex stability and ionic conditions on the periodicity of the NS3 unwinding cycle. The kinetic step size for NS3 unwinding was examined on diverse substrate sequences. The kinetic step size (16 bp/step) was found to be independent of RNA duplex stability and composition, but it exhibited strong dependence on monovalent salt concentration, decreasing to approximately 11 bp/step at low [NaCl]. We addressed this behavior by analyzing the oligomeric state of NS3 at various salt concentrations. Whereas only NS3 oligomers are capable of processive unwinding, we found that monomeric NS3 is an active helicase that unwinds with low processivity. We demonstrate that low salt conditions enhance unwinding by monomeric NS3, which is likely to account for the reduction in apparent step size under low salt conditions. Based on results reported here, as well as available structural and single molecule data, we present an unwinding mechanism that addresses the apparent periodicity of NS3 unwinding, the magnitude of the step size, and that integrates the various stepwise motions observed for NS3. We propose that the large kinetic step size of NS3 unwinding reflects a delayed, periodic release of the separated RNA product strand from a secondary binding site that is located in the NTPase domain (Domain II) of NS3. These findings suggest that the mechanism of product release represents an important and unexplored feature of helicase mechanism.

PMID:
19010782
PMCID:
PMC2629105
DOI:
10.1074/jbc.M805460200
[Indexed for MEDLINE]
Free PMC Article

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