NS3 Helicase from the Hepatitis C Virus Can Function as a Monomer or Oligomer Depending on Enzyme and Substrate Concentrations
2008; Elsevier BV; Volume: 284; Issue: 8 Linguagem: Inglês
10.1074/jbc.m805540200
ISSN1083-351X
AutoresThomas Jennings, Samuel G. Mackintosh, Melody K. Harrison, Deniz Sikora, Bartek Sikora, Bhuvanesh Dave, Alan J. Tackett, Craig E. Cameron, Kevin D. Raney,
Tópico(s)Biochemical and Molecular Research
ResumoHepatitis 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. 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 (1Delagoutte E. von Hippel P.H. Q. Rev. Biophys. 2002; 35: 431-478Crossref PubMed Scopus (146) Google Scholar, 2Lohman T.M. Bjornson K.P. Annu. Rev. Biochem. 1996; 65: 169-214Crossref PubMed Scopus (670) Google Scholar, 3Patel S.S. Picha K.M. Annu. Rev. Biochem. 2000; 69: 651-697Crossref PubMed Scopus (462) Google Scholar, 4Soultanas P. Wigley D.B. Trends Biochem. Sci. 2001; 26: 47-54Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 5von Hippel P.H. Delagoutte E. Cell. 2001; 104: 177-190Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). 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 (6Dong F. Gogol E.P. von Hippel P.H. J. Biol. Chem. 1995; 270: 7462-7473Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 7Jezewska M.J. Rajendran S. Bujalowska D. Bujalowski W. J. Biol. Chem. 1998; 273: 10515-10529Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Indeed, a large number of helicases form and function as hexameric structures (3Patel S.S. Picha K.M. Annu. Rev. Biochem. 2000; 69: 651-697Crossref PubMed Scopus (462) Google Scholar). PcrA, a Gram-positive bacterial helicase, translocates on single-stranded DNA as a monomer (8Dillingham M.S. Wigley D.B. 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Virol. 1995; 69: 1769-1777Crossref PubMed Google Scholar). However, the activities are interdependent, since recently it was shown that the NS3 helicase domain stimulates the serine protease activity (19Beran R.K. Pyle A.M. J. Biol. Chem. 2008; 283: 29929-29937Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Both protease and helicase activities are required for viral replication (20Kolykhalov A.A. Mihalik K. Feinstone S.M. Rice C.M. J. Virol. 2000; 74: 2046-2051Crossref PubMed Scopus (563) Google Scholar). NS3 binds to a protein co-factor, NS4A, which activates the protease activity (21Gallinari P. Paolini C. Brennan D. Nardi C. Steinkuhler C. De F.R. Biochemistry. 1999; 38: 5620-5632Crossref PubMed Scopus (65) Google Scholar). Crystal structures of NS3 and NS3 helicase domain (NS3h) have been reported (22Kang L.W. Cho H.S. Cha S.S. Chung K.M. Back S.H. Jang S.K. Oh B.H. Acta Crystallogr. Sect. D Biol. 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Nature. 2004; 430: 476-480Crossref PubMed Scopus (120) Google Scholar, 33Tackett A.J. Chen Y. Cameron C.E. Raney K.D. J. Biol. Chem. 2005; 280: 10797-10806Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Dumont et al. (29Dumont S. Cheng W. Serebrov V. Beran R.K. Tinoco Jr., I. Pyle A.M. Bustamante C. Nature. 2006; 439: 105-108Crossref PubMed Scopus (299) Google Scholar) have reported that NS3 is active as a monomer with an 11-bp kinetic step size in optical trap unwinding experiments. Serebrov and Pyle (32Serebrov V. Pyle A.M. Nature. 2004; 430: 476-480Crossref PubMed Scopus (120) Google Scholar) have reported that the active form of NS3 helicase is a dimer with an 18-bp kinetic step size through the use of a unique set of randomly nicked substrates. Tackett et al. (33Tackett A.J. Chen Y. Cameron C.E. Raney K.D. J. Biol. Chem. 2005; 280: 10797-10806Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) have reported that multiple molecules of NS3 bind to a single DNA duplex and are required for optimal unwinding. Most recently, Sikora et al. (34Sikora B. Chen Y. Lichti C.F. Harrison M.K. Jennings T.A. Tang Y. Tackett A.J. Jordan J.B. Sakon J. Cameron C.E. Raney K.D. J. Biol. Chem. 2008; 283: 11516-11525Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) have shown that NS3 can form oligomeric structures and that the enzyme's optimal DNA unwinding activity correlates with formation of those structures. The question arises as to whether the monomeric form of NS3 also has DNA unwinding activity and, if so, how it relates to the oligomeric forms of the enzyme. Several kinetic and physical methods exist for determining the oligomeric state of helicase enzymes. Poisoning the activity of an ensemble with inactive protein has been used to illustrate interactions or the lack of interactions among protein monomers for the NS3 helicase domain (35Levin M.K. Patel S.S. J. Biol. Chem. 1999; 274: 31839-31846Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 36Preugschat F. Danger D.P. Carter III, L.H. Davis R.G. Porter D.J. Biochemistry. 2000; 39: 5174-5183Crossref PubMed Scopus (28) Google Scholar). However, the study demonstrating interactions based on a dominant-negative phenotype has been reinterpreted as a functionally cooperative interaction that does not require canonical protein-protein interactions (37Levin M.K. Wang Y.H. Patel S.S. J. Biol. Chem. 2004; 279: 26005-26012Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). In order to address the question of whether oligomerization or cooperativity is required for DNA unwinding by hepatitis C virus (HCV) NS3, a series of experiments were performed to measure NS3 unwinding activity under a variety of conditions. Previous experiments were designed to maximize processivity by using a vast excess of enzyme over nucleic acid. In the current report, NS3 unwinding activity was measured over a range of enzyme concentrations and enzyme-to-substrate ratios in order to examine the activity under conditions that disfavor binding of multiple helicase molecules to a single substrate molecule. Such conditions should allow discernment of helicase activity that can be attributed to monomeric as compared with oligomeric forms of this enzyme. Materials—HEPES, EDTA, β-mercaptoethanol (βME), 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. Sephadex G-25, phosphoenolpyruvate kinase/lactate dehydrogenase, NADH, ATP, and phosphoenolpyruvate were from Sigma. Poly(U) was purchased from Amersham Biosciences. DNA oligonucleotides were from Integrated DNA Technologies and purified by preparative gel electrophoresis (38Morris P.D. Tackett A.J. Raney K.D. Methods. 2001; 23: 149-159Crossref PubMed Scopus (29) Google Scholar). [γ-32P]ATP was purchased from PerkinElmer Life Sciences. T4 polynucleotide kinase was obtained from New England Biolabs. Recombinant full-length NS3, NS3(D290A), and NS3-tetra-Cys were derived from 1b replicon consensus sequence and expressed and purified as SUMO fusion proteins. 3D. Sikora, B. Sikora, and K. D. Raney, manuscript in preparation. 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 (39Tackett A.J. Wei L. Cameron C.E. Raney K.D. Nucleic Acids Res. 2001; 29: 565-572Crossref PubMed Scopus (62) Google Scholar). 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). NS3-tetra-Cys and FlAsH-EDT2 (Invitrogen) were incubated at 4 °C for 5 h with gentle agitation. After dialysis to remove free dye, ∼85% of the protein was found to have bound the dye. The resulting labeled protein was referred to NS3-FlAsH. 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 βME, 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. The sequence of the 45-nt oligonucleotide was 5′-GACTGACGCTAGGCTGACAGGACGTACTACT15-3′. The sequence of the 30-nt complementary strand was 5′-GTAGTACGTCCTGTCAGCCTAGCGTCAGTC-3′. The unwinding reaction was initiated by the addition of 5 mm ATP, 10 mm MgCl2, and a 30-fold excess of DNA trap to bind the displaced strand and prevent reannealing to the radiolabeled product. At appropriate time points, a 10-μl aliquot of the reaction mixture was transferred to a centrifuge tube containing 200 mm EDTA, 0.7% SDS, 0.1% bromphenol blue, 0.1% xylene cyanol, and 6% glycerol. The double- and single-stranded DNA were resolved via native 20% polyacrylamide gel. The radiolabeled substrate and product were detected using a PhosphorImager (Amersham Biosciences). Quantitation was performed with ImageQuant software (GE Healthcare), and the ratio of single- to double-stranded DNA was plotted as a function of time. Data were fit to a straight line using Kaleidagraph (Synergy Software, Reading, PA). Identical experiments were performed at 100 and 500 nm NS3wt incubated with varying amounts of ATPase-deficient NS3(D290A) with 100 nm and 1.25 μm substrate, respectively. 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 βME, and 0.1 mg/ml BSA. The protein(s) was co-incubated for 90 min at 37 °C. Substrate was then incubated with the protein(s) for 5 min. Reaction components were incubated at 37 °C using a circulating water bath. The unwinding reaction was initiated by the rapid addition of 5 mm ATP, 10 mm MgCl2 (unless otherwise specified), 30-fold excess DNA trap, and 10-fold excess poly(U) protein trap using an RQF-3 Rapid Quench Flow instrument (KinTek Corp., Austin, TX). The reaction was quenched after 0.1–30 s by adding 200 mm EDTA and 0.7% SDS. Bromphenol blue (0.1%), 0.1% xylene cyanol, and 6% glycerol were added to each, and the double- and single-stranded DNA were resolved via native 20% polyacrylamide gel. The radiolabeled substrate and product were detected using a PhosphorImager, and quantitation was performed with ImageQuant software (GE Healthcare). Data were converted to concentration of ssDNA product as described (33Tackett A.J. Chen Y. Cameron C.E. Raney K.D. J. Biol. Chem. 2005; 280: 10797-10806Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Fluorescence Anisotropy Assay—Experiments were performed at 37 °C using a PerkinElmer Life Sciences Victor3V 1420 multilabel counter. Measurements were integrated over a 0.2-s detection period. The instrument lamp energy was at the maximum setting, and filters were set to 485 nm for excitation and 535 nm for emission. The polarizing aperture was set to normal, and the excitation aperture was set to 4 mm. NS3-tetra-Cys was labeled with FlAsH and prepared as described (40Jennings T.A. Chen Y. Sikora D. Harrison M.K. Sikora B. Huang L. Jankowsky E. Fairman M.E. Cameron C.E. Raney K.D. Biochemistry. 2008; 47: 1126-1135Crossref PubMed Scopus (36) Google Scholar). The labeled protein was referred to as NS3-FlAsH. 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 βME, and 0.1 mg/ml BSA. Triplicate samples were incubated at 37 °C for 90 min prior to measuring fluorescence polarization. Data were plotted as the average of three independent experiments with S.D. values. Fluorescence Resonance Energy Transfer Assay—Measurements were made using a SLM Aminco-Bowman Series 2 luminescence spectrometer. The temperature was regulated at 37 °C with a circulating water bath. The excitation wavelength was set to 550 nm with a band pass of 0.5 nm. The emission wavelength was set to 668 nm with a band pass of 8 nm. Buffer consisted of 25 mm MOPS, pH 7.0, 10 mm NaCl, 0.1 mm EDTA, 1 mm βME, and 0.1 mg/ml BSA. 5′-Cy3-labeled and 3′-Cy5-labeled, noncomplementary, 8-base oligonucleotides were added to the buffer to a final concentration of 500 nm each. The sequences for the probes were 5′-GTCACACT-Cy5–3′ and 5′-Cy3-AGCATCAG-3′. NS3wt or NS3h was then titrated into solution, and the intensity of emission at 668 nm was detected. Data for NS3wt were plotted as the average of three experiments with S.D. values. The experiment was repeated with each oligonucleotide probe at 2.5 μm, and NS3wt was titrated into the solution. Fluorescence Anisotropy of NS3-FlAsH with NS3wt or NS3h—Previous work has indicated that NS3 interacts with itself. Results have indicated a dimeric form of the enzyme (30Khu Y.L. Koh E. Lim S.P. Tan Y.H. Brenner S. Lim S.G. Hong W.J. Goh P.Y. J. Virol. 2001; 75: 205-214Crossref PubMed Scopus (34) Google Scholar, 32Serebrov V. Pyle A.M. Nature. 2004; 430: 476-480Crossref PubMed Scopus (120) Google Scholar), whereas other studies have suggested that larger oligomers can also form (34Sikora B. Chen Y. Lichti C.F. Harrison M.K. Jennings T.A. Tang Y. Tackett A.J. Jordan J.B. Sakon J. Cameron C.E. Raney K.D. J. Biol. Chem. 2008; 283: 11516-11525Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). As a biophysical method of investigating the interaction of NS3 with itself, a fluorescence anisotropy assay was employed. Fluorescence anisotropy is a technique that reports on the size and shape of a species in solution (41Lundblad J.R. Laurance M. Goodman R.H. Mol. Endocrinol. 1996; 10: 607-612Crossref PubMed Scopus (216) Google Scholar). If the size of a species increases upon interactions with a binding partner, then the degree of fluorescence polarization increases. Conversely, if a species is broken into smaller units upon mixing, then polarization decreases. To conduct these experiments, we prepared a variant of NS3 containing a peptide (Cys-Cys-Pro-Gly-Cys-Cys) on the C terminus (NS3-tetra-Cys) that binds tightly to a fluorescein derivative termed FlAsH (42Griffin B.A. Adams S.R. Tsien R.Y. Science. 1998; 281: 269-272Crossref PubMed Scopus (1280) Google Scholar, 43Griffin B.A. Adams S.R. Jones J. Tsien R.Y. Methods Enzymol. 2000; 327: 565-578Crossref PubMed Scopus (215) Google Scholar). The labeled protein, NS3-FlAsH, was found to behave similarly to wild-type NS3 with regard to DNA unwinding (supplemental Fig. 1). NS3-FlAsH was titrated with NS3wt and NS3h in unwinding assay buffer (Fig. 1). 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 NS3h. Fitting the data to the quadratic equation results in an apparent Kd value of 236 ± 52 nm. This result provides direct support for NS3 oligomerization in solution and highlights the importance of the protease domain in mediating NS3-NS3 interactions, as was previously reported (30Khu Y.L. Koh E. Lim S.P. Tan Y.H. Brenner S. Lim S.G. Hong W.J. Goh P.Y. J. Virol. 2001; 75: 205-214Crossref PubMed Scopus (34) Google Scholar). However, one consideration is that the observed equilibrium dissociation constant in Fig. 1 may be reflective of multiple equilibria, including monomers associating to form dimers, dimers associating to form tetramers, and so on, in light of previous studies indicating that quaternary structures larger than dimers are likely to exist at concentrations of NS3 of ≥1 μm (34Sikora B. Chen Y. Lichti C.F. Harrison M.K. Jennings T.A. Tang Y. Tackett A.J. Jordan J.B. Sakon J. Cameron C.E. Raney K.D. J. Biol. Chem. 2008; 283: 11516-11525Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). NS3 Behaves as an Oligomer in Solution That Is Capable of Binding Multiple Nucleic Acid Strands—To explore whether NS3 oligomers can bind to DNA, a fluorescence resonance energy transfer (FRET) experiment was designed to simultaneously report on the oligomerization of the protein as well as the number of active DNA binding sites within the oligomer. Binding of DNA to NS3 as a function of NS3 concentration was examined by using oligonucleotides that were designed to bind in a 1:1 stoichiometry with NS3. The nucleic acid binding site of NS3 has previously been shown to accommodate 8 nucleotides (25Kim J.L. Morgenstern K.A. Griffith J.P. Dwyer M.D. Thomson J.A. Murcko M.A. Lin C. Caron P.R. Structure. 1998; 6: 89-100Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar). Therefore, Cy3- and Cy5-labeled 8-mers were used as probes to minimize the possibility of multiple NS3 monomers binding to an individual nucleic acid sequence. The Cy3 and Cy5 FRET pair was chosen based on their successful application in protein-nucleic acid measurements. The Förster radius for this pair has been determined to be 61–65 Å (44Rasnik I. Myong S. Cheng W. Lohman T.M. Ha T. J. Mol. Biol. 2004; 336: 395-408Crossref PubMed Scopus (135) Google Scholar). Hence, upon titration of the mixture of oligonucleotides with NS3, FRET should be observed if the oligonucleotides bind to adjacent sites within an oligomeric form of NS3 as depicted in Fig. 2A. The concentration of each nucleic acid was initially set at 500 nm in order to be above the equilibrium dissociation constant for ssDNA binding under these conditions (7 nm; supplemental Fig. 2). The raw emission spectra for the oligonucleotide probes alone and in the presence of 1.5 μm NS3 are shown in Fig. 2B. The emission from the Cy3 fluorophore is reduced in the spectrum generated from the sample containing NS3 with a concomitant increase in the signal from the Cy5 label, as expected for a FRET signal. The emission was measured as a function of increasing NS3 concentration and is plotted in Fig. 2C. Interestingly, the FRET signal reached a maximum at a ∼1:1 ratio of protein to nucleic acid strands, which indicates that all of the protein is capable of binding nucleic acid substrate. Additionally, NS3h, which does not display the oligomeric characteristics of full-length NS3, did not exhibit an increase in FRET acceptor signal as protein was titrated into a solution containing labeled oligonucleotides. The sigmoidal shape of the curve in Fig. 2C suggests that positive cooperativity occurs due to protein-protein interactions, protein-nucleic acid interactions, or both. If protein-protein interactions are solely responsible for the sigmoidal shape, then repeating the titration at higher nucleic acid concentration (and hence higher protein concentration) may reduce the sigmoidicity. The concentration of each oligonucleotide probe was increased to 2.5 μm, and the titration was repeated. As with the experiment performed at lower DNA concentration, the shape of the binding curve was sigmoidal, and the FRET signal reached a maximum point when the nucleic acid strand/protein ratio was 1:1 (Fig. 2D). Overlaying the scaled data from Fig. 2, C and D (Fig. 2E) shows that both curves demonstrate a very similar sigmoidal increase in signal, suggesting that the initial phase of the curves may due to a threshold that must be exceeded for the development of FRET signal. Thus, the equilbria that are responsible for the shape of the FRET signal as a function of NS3 concentration are likely to involve protein-protein and protein-nucleic acid interactions. There are several clear results from this experiment: 1) NS3 forms oligomeric species consisting of, at a minimum, dimers in the presence of nucleic acids; 2) adjacent monomers within the oligomeric structure are capable of binding nucleic acid substrates; and 3) all of the protein is capable of binding to DNA even after oligomerization. DNA Unwinding at Varying Concentrations of NS3 Indicates no Increase in Specific Activity under Multiple Turnover Conditions— Formation of NS3 oligomeric structures has been correlated with increasing DNA and RNA unwinding activity by showing that the burst amplitude for product formation increases with increasing enzyme concentration (34Sikora B. Chen Y. Lichti C.F. Harrison M.K. Jennings T.A. Tang Y. Tackett A.J. Jordan J.B. Sakon J. Cameron C.E. Raney K.D. J. Biol. Chem. 2008; 283: 11516-11525Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The reported experiments were conducted under single turnover conditions in which the enzyme concentration was similar to or greater than the DNA substrate concentration. The results indicated that if a monomeric form of NS3 is active, then its processivity is relatively low compared with oligomeric forms. DNA unwinding can be measured under multiple turnover conditions in which the substrate concentration is similar to or greater than the enzyme concentration. Multiple turnover conditions effectively increase the "signal" for measuring DNA unwinding by allowing multiple opportunities for unwinding to occur. If NS3 must oligomerize to function, then the specific activity of the enzyme should increase as the enzyme concentration increases. To determine if the active species of NS3 changes with a dependence on enzyme concentration, we measured unwinding of a DNA substrate under multiple turnover conditions over a range of enzyme concentrations, both below and above the apparent Kd value observed by anisotropy. If NS3-NS3 interactions are necessary for the observed DNA unwinding activity, then an increase in specific activity with increasing enzyme concentration should be observed, as long as DNA unwinding is rate-limiting in the reaction. An increase in the unwinding rate with increasin
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