NS3 Helicase from the Hepatitis C Virus Can Function as a Monomer or Oligomer Depending on Enzyme and Substrate Concentrations

Thomas A Jennings,Samuel G Mackintosh,Melody K Harrison,Deniz Sikora,Bartek Sikora,Bhuvanesh Dave,Alan J Tackett,Craig E Cameron,Kevin D Raney

doi:10.1074/jbc.m805540200

Abstract

Hepatitis C virus NS3 helicase can unwind double-stranded DNA and RNA and has been proposed to form oligomeric structures. Here we examine the DNA unwinding activity of monomeric NS3. Oligomerization was measured by preparing a fluorescently labeled form of NS3, which was titrated with unlabeled NS3, resulting in a hyperbolic increase in fluorescence anisotropy and providing an apparent equilibrium dissociation constant of 236 nm. To evaluate the DNA binding activity of individual subunits within NS3 oligomers, two oligonucleotides were labeled with fluorescent donor or acceptor molecules and then titrated with NS3. Upon the addition of increasing concentrations of NS3, fluorescence energy transfer was observed, which reached a plateau at a 1:1 ratio of NS3 to oligonucleotides, indicating that each subunit within the oligomeric form of NS3 binds to DNA. DNA unwinding was measured under multiple turnover conditions with increasing concentrations of NS3; however, no increase in specific activity was observed, even at enzyme concentrations greater than the apparent dissociation constant for oligomerization. An ATPase-deficient form of NS3, NS3(D290A), was prepared to explore the functional consequences of oligomerization. Under single turnover conditions in the presence of excess concentration of NS3 relative to DNA, NS3(D290A) exhibited a dominant negative effect. However, under multiple turnover conditions in which DNA concentration was in excess to enzyme concentration, NS3(D290A) did not exhibit a dominant negative effect. Taken together, these data support a model in which monomeric forms of NS3 are active. Oligomerization of NS3 occurs, but subunits can function independently or cooperatively, dependent upon the relative concentration of the DNA.

Highlights

Transcription, translation, repair, and recombination [1,2,3,4,5]
NS3 helicase has a 3Ј to 5Ј directional bias in unwinding, it is capable of unwinding both RNA and DNA double helices, and it binds more avidly to U-rich or dT-rich nucleic acid substrates (26 –28)
The results show an increase in anisotropy when the labeled protein is mixed with NS3wt, whereas little or no change is noted when it is mixed with NS3 helicase domain (NS3h)

Summary

EXPERIMENTAL PROCEDURES

Materials—HEPES, EDTA, ␤-mercaptoethanol (␤ME), SDS, MOPS, Tris, NaCl, Na4EDTA, SDS, BSA, HEPES, acrylamide, bisacrylamide, MgCl2, KOH, ATP, formamide, xylene cyanole, bromophenol blue, urea, glycerol, and MgCl2 were purchased from Fisher. Multiple Turnover DNA Unwinding—NS3 was prepared in 25 mM MOPS (pH 7.0), 10 mM NaCl, 0.1 mM EDTA (pH 8.0), 2 mM ␤ME, and 0.1 mg/ml BSA. DNA substrate (15 nt/30 bp, the longer strand radiolabeled with 32P) was added to 100 nM, and the mixture was incubated at 37 °C for 5 min. Single Turnover DNA Unwinding—NS3 (250 nM NS3wt with variable NS3(D290A)) and DNA substrate (2 nM 15 nt/30 bp, the longer strand radiolabeled with 32P) were prepared in 25 mM MOPS (pH 7.0), 10 mM NaCl, 0.1 mM EDTA (pH 8.0), 2 mM ␤ME, and 0.1 mg/ml BSA. A solution of NS3-FlAsH (50 nM) was titrated against an increasing concentration of NS3wt or NS3h in buffer containing 25 mM MOPS, pH 7.0, 10 mM NaCl, 0.1 mM EDTA, 1 mM ␤ME, and 0.1 mg/ml BSA. The experiment was repeated with each oligonucleotide probe at 2.5 ␮M, and NS3wt was titrated into the solution

RESULTS

70 Ϯ 2 60 Ϯ 1 42 Ϯ 2 32 Ϯ 3 16 Ϯ 3

DISCUSSION