Unwinding of nucleic acids by HCV NS3 helicase is sensitive to the structure of the duplex

Tackett, Alan J.; Wei, Lai; Cameron, Craig Ε.; Raney, Kevin D.

doi:10.1093/nar/29.2.565

Cited by 68 publications

(76 citation statements)

References 33 publications

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“…3A). The ''two points of contact'' model presented here is consistent with the inchworming mechanism previously proposed for this enzyme (4) and also with the sensitivity of NS3 helicase activity to the structure of the duplex from bulk studies (25).…”

Section: Discussionsupporting

confidence: 88%

NS3 helicase actively separates RNA strands and senses sequence barriers ahead of the opening fork

Cheng¹,

Dumont

Tinoco³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

149

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RNA helicases regulate virtually all RNA-dependent cellular processes. Although much is known about helicase structures, very little is known about how they deal with barriers in RNA and the factors that affect their processivity. The hepatitis C virus encodes NS3, an RNA helicase that is essential for viral RNA replication. We have used optical tweezers to determine at the single-molecule level how the local stability of the RNA substrate affects the enzyme rate of strand separation, whether separation occurs by an active or a passive mechanism, and whether processivity is affected. We show that sequence barriers in RNA modulate NS3 activity. NS3 processivity depends on barriers ahead of the opening fork. Our results rule out a model where NS3 passively waits for the thermal fraying of double-stranded RNA. Instead, we find that NS3 destabilizes the duplex before separating the strands. Failure to do so before a strong barrier leads to helicase dissociation and limits the processivity of the enzyme.hairpin ͉ molecular motors ͉ optical tweezers ͉ processivity A wide range of RNA metabolic activities (1) require the breaking of base pairs in double-stranded RNA (dsRNA) by RNA helicases. Despite recent progress in the study of these motor proteins (2-4), the physical mechanisms by which they move and catalyze the strand separation are not well understood. Helicase models ranging from a pure Brownian ratchet to a pure power stroke action have been discussed (5-8), but experimental data to support them for RNA helicases have been lacking. The hepatitis C virus NS3 protein is a superfamily 2, 3Ј to 5Ј RNA helicase (9) known to be essential for virus replication and, thus, an antiviral drug target (10). Recently, NS3 has been the subject of single-molecule manipulation studies (4), which revealed that NS3 makes steps of 11 bp each made up of three substeps (4). RNA molecules contain various kinetic barriers to mechanical unfolding (11). How these barriers influence the activity of motor proteins that work on RNA will depend on the mechanism of the motor. To probe the molecular mechanism of NS3 unwinding activity, we have designed and characterized RNA molecules with various mechanical unfolding barriers and used single-molecule methods to investigate how these barriers affect the velocity, pausing, and processivity of the helicase in real time. ResultsDesign and Characterization of RNA Hairpin Substrates. We first designed two RNA hairpin substrates that terminate both on a tetraloop (Fig. 1A) to monitor the response of a single NS3 helicase to weak and strong sequence barriers. Substrate RNA-AG has 30 A⅐U pairs followed by 30 G⅐C pairs; RNA-GA has the A⅐U and G⅐C sequences interchanged. The sequences within A⅐U and G⅐C regions were chosen to minimize the formation of alternative secondary structures other than the desired 60-bp hairpin. In our experiment, a single RNA hairpin was attached between a microsphere in an optical trap and a microsphere placed on the end of a micropipette through hybrid RNA-DNA handles to se...

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Section: Discussionsupporting

confidence: 88%

NS3 helicase actively separates RNA strands and senses sequence barriers ahead of the opening fork

Cheng¹,

Dumont

Tinoco³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

149

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“…This rate is comparable or even faster than the reported nucleic acid unwinding rates of either the helicase domain or the full-length protease-helicase with or without the His tag (10,11,36). It was also shown recently that the DNA unwinding activity of the helicase domain with C-terminal or N-terminal His tag was similar (11).…”

Section: Resultssupporting

confidence: 62%

The Functional Interaction of the Hepatitis C Virus Helicase Molecules Is Responsible for Unwinding Processivity

Levin

Wang

Patel

2004

Journal of Biological Chemistry

116

134

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Although helicases participate in virtually every cellular process involving nucleic acids, the details of their mechanism including the role of interaction between the subunits remains unclear. Here we study the unwinding kinetics of the helicase from hepatitis C virus using DNA substrates with a range of tail and duplex lengths. The binding of the helicase to the substrates was characterized by electron microscopy and fluorimetric titrations. Depending on the length of the ssDNA tail, one or more helicase molecules can be loaded on the DNA. Unwinding was measured under single-turnover conditions, and the results show that a monomer is active on short duplexes yet multiple molecules are needed to unwind long duplexes. Thus, increasing the ssDNA tail length increases the unwinding efficiency. The unwinding kinetics was modeled as a stepwise process performed by single or multiple helicase molecules. The model programmed in MATLAB was used for global fitting of the kinetics, yielding values for the rate of unwinding, processivity, cooperativity, step size, and occlusion site. The results indicate that a single hepatitis C virus helicase molecule unwinds DNA with a low processivity. The multiple helicase molecules present on the DNA substrate show functional cooperativity and unwind with greater efficiency, although they bind and release the substrate non-cooperatively, and the ATPase cycle of the helicase molecules is not coordinated. The functional interaction model explains the efficient unwinding by multiple helicases and is generally applicable.Helicases are motor proteins that translocate along DNA or RNA using ATP hydrolysis. The translocation activity is required for strand separation of the duplex nucleic acids, the elimination of secondary structure in RNA, and to dissociate proteins bound to the nucleic acids (1-4). The exact mechanism of translocation and nucleic acid strand separation is not known for any helicase. However, unwinding is believed to be a stepwise process that among other things may require interaction between helicase molecules. In this paper we use single turnover unwinding kinetics experiments as well as numerical modeling to investigate the role of subunit interactions during unwinding by the helicase from hepatitis C virus.Hepatitis C virus (HCV) 1 contains a single stranded RNA genome that codes for a polyprotein, which is cleaved into structural and nonstructural (NS) proteins. The NS3 protein of the HCV is both a helicase and a protease. The crystal structure of NS3 shows two loosely connected domains (5). The helicase activity resides on the C-terminal domain that constitutes ϳ450 C-terminal amino acid residues and the protease activity on the N-terminal domain. The NS3 protease is tightly associated with its essential co-factor NS4A, which is predicted to be membrane-bound. The NS3 helicase is, therefore, tethered to the endoplasmic reticulum membrane in vivo. The protease and helicase activities appear to be independent as these domains can be expressed separately in Escheric...

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“…3 The sumoylation was removed during the purification process by incubating the crudely purified protein with Ulp 1 protease. NS3 helicase domain (NS3h) derived from the same Con 1b sequence was expressed and purified as previously described (39). A form of NS3, NS3-tetra-Cys, was engineered to have a tetracysteine cassette (Cys-Cys-Pro-Gly-Cys-Cys) located at the C terminus of the protein by using the QuikChange mutagenesis kit (Stratagene).…”

Section: Methodsmentioning

confidence: 99%

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

Jennings

Mackintosh

Harrison

et al. 2009

Journal of Biological Chemistry

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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.Helicases are ubiquitous enzymes required for virtually all cellular processes involving nucleic acids, including replication, transcription, translation, repair, and recombination (1-5). These enzymes catalyze unwinding of double-stranded DNA or RNA by converting chemical energy from ATP hydrolysis into mechanical energy for nucleic acid strand separation. However, there is considerable variability in the quaternary structure of the active forms of helicases. Some helicases function effectively in unwinding activities as monomers, whereas others are active as dimers or oligomers. For example, bacteriophage T4 gp41 helicase and Escherichia coli DnaB helicase form hexameric structures that encircle and sequester single-stranded DNA (6, 7). Indeed, a large number of helicases form and function as hexameric structures (3). PcrA, a Gram-positive bacterial helicase, translocates on single-stranded DNA as a monomer (8) and has been proposed to unwind double-stranded DNA as a monomer (9). Bacteriophage T4 Dda helicase has activity in the monomeric form (10) but demonstrates more efficient unwinding activity under conditions where multiple helicase molecules bind a given substrate molecule (11). It has been shown that E. coli Rep helicase unwinds DNA as a dimer (12). UvrD has...

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Unwinding of nucleic acids by HCV NS3 helicase is sensitive to the structure of the duplex

Cited by 68 publications

References 33 publications

NS3 helicase actively separates RNA strands and senses sequence barriers ahead of the opening fork

NS3 helicase actively separates RNA strands and senses sequence barriers ahead of the opening fork

The Functional Interaction of the Hepatitis C Virus Helicase Molecules Is Responsible for Unwinding Processivity

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

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