The Functional Interaction of the Hepatitis C Virus Helicase Molecules Is Responsible for Unwinding Processivity
2004; Elsevier BV; Volume: 279; Issue: 25 Linguagem: Inglês
10.1074/jbc.m403257200
ISSN1083-351X
AutoresMikhail K. Levin, Yuh-Hwa Wang, Smita S. Patel,
Tópico(s)Viral Infections and Immunology Research
ResumoAlthough 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. 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. The functional interaction of the hepatitis C virus helicase molecules is responsible for unwinding processivity. Vol. 279 (2004) 26005–26012Journal of Biological ChemistryVol. 279Issue 40PreviewWe use the incomplete gamma function (Equation 4) to model the multistep kinetics of helicase DNA unwinding. We wish to make a clarification that this method was developed independently in our laboratory but was first described in Lucius et al. (Lucius, A. L., Vindigni, A., Gregorian, R., Ali, J. A., Taylor, A. F., Smith, G. R., and Lohman, T. M. (2002) J. Mol. Biol.324, 409–428 and Lucius, A. L., Maluf, N. K., Fischer, C. J., and Lohman, T. M. (2003) Biophys. J.85, 2224–2239). These papers were referenced as (37) and (35), respectively, in conjunction with the modeling of the helicase kinetics. Full-Text PDF Open Access 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 (1Patel S.S. Picha K.M. Annu. Rev. Biochem. 2000; 69: 651-697Crossref PubMed Scopus (462) Google Scholar, 2Lohman T.M. Bjornson K.P. Annu. Rev. Biochem. 1996; 65: 169-214Crossref PubMed Scopus (671) Google Scholar, 3Levin M.K. Patel S.S. Schliwa M. Molecular Motors. Wiley-VCH Verlag GmbH, Weinheim Germany2003: 179-198Google Scholar, 4Matson S.W. Bean D.W. George J.W. BioEssays. 1994; 16: 13-22Crossref PubMed Scopus (270) Google Scholar). 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) 1The abbreviations used are: HCV, hepatitis C virus; ds, double-stranded; ss, single-stranded; MOPS, 3[N-morpholino]propanesulfonic acid; NS3h, helicase domain of HCV non-structural protein 3; NC, nitrocellulose; nt, nucleotide(s); TLC, thin layer chromatography; NS protein, nonstructural protein; EM, electron microscopy. 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 (5Yao N. Reichert P. Taremi S.S. Prosise W.W. Weber P.C. Structure Fold. Des. 1999; 7: 1353-1363Abstract Full Text Full Text PDF Scopus (369) Google Scholar). The helicase activity resides on the C-terminal domain that constitutes ∼450 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 Escherichia coli while retaining their full activity (6Gwack Y. Kim D.W. Han J.H. Choe J. Biochem. Biophys. Res. Commun. 1996; 225: 654-659Crossref PubMed Scopus (131) Google Scholar, 7Han D.S. Hahm B. Rho H.M. Jang S.K. J. Gen. 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There is no consensus in the literature about the effect of the protease-NS4A on the helicase activity (10Pang P.S. Jankowsky E. Planet P.J. Pyle A.M. EMBO J. 2002; 21: 1168-1176Crossref PubMed Scopus (205) Google Scholar, 11Frick D.N. Rypma R.S. Lam A.M. Gu B. J. Biol. Chem. 2004; 279: 1269-1280Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 13Gallinari P. Brennan D. Nardi C. Brunetti M. Tomei L. Steinkühler C. De Francesco R. J. Virol. 1998; 72: 6758-6769Crossref PubMed Google Scholar, 14Howe A.Y. Chase R. Taremi S.S. Risano C. Beyer B. Malcolm B. Lau J.Y. Protein Sci. 1999; 8: 1332-1341Crossref PubMed Scopus (51) Google Scholar, 15Gallinari P. Paolini C. Brennan D. Nardi C. Steinkuhler C. De Francesco R. Biochemistry. 1999; 38: 5620-5632Crossref PubMed Scopus (65) Google Scholar, 16Kuang W.F. Lin Y.C. Jean F. Huang Y.W. Tai C.L. Chen D.S. Chen P.J. Hwang L.H. Biochem. Biophys. Res. Commun. 2004; 317: 211-217Crossref PubMed Scopus (42) Google Scholar). The virus requires both helicase and protease functions, but it is not clear if the protease-NS4A activates or inhibits the helicase. We have previously (17Levin M.K. Patel S.S. J. Biol. Chem. 1999; 274: 31839-31846Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) characterized the helicase activity of NS3h under multiple turnover conditions using a partially duplex DNA substrate that contained a 10-nt-long ssDNA tail and a 33-bp-long duplex. Under multiple turnover conditions, the helicase rate depended on the NS3h concentration, indicating that the observed rate of unwinding is limited by the rebinding of free NS3h to DNA. We also observed that the unwinding rate decreased sharply upon the addition of small amounts of an ATPase-deficient NS3h mutant, but the amplitude of unwinding changed little. This led us to conclude that many NS3h molecules participate in the unwinding of each DNA substrate. It is not clear, however, whether a single molecule of the HCV helicase can unwind DNA and, in cases where multiple helicase molecules are required, whether they work cooperatively. This question is relevant to other helicases as well. For the HCV helicase, most of the evidence thus far indicates that it does not form a stable oligomer in solution. Gel filtration chromatography, ultra centrifugation and x-ray crystallography show that NS3h is a monomer (17Levin M.K. Patel S.S. J. Biol. Chem. 1999; 274: 31839-31846Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 18Kim 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, 19Porter D.J.T. Short S.A. Hanlon M.H. Preugschat F. Wilson J.E. Willard Jr., D.H. Consler T.G. J. Biol. Chem. 1998; 273: 18906-18914Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Protein-protein cross-linking of NS3h occurs inefficiently, and the addition of an ATPase-deficient mutant to wild type NS3h does not inhibit its ATPase activity (17Levin M.K. Patel S.S. J. Biol. Chem. 1999; 274: 31839-31846Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Recently, we showed that the NS3h ATPase activity is independent of NS3h concentration when measured in the presence of a non-ionic detergent (17Levin M.K. Patel S.S. J. Biol. Chem. 1999; 274: 31839-31846Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Fluorimetric titration experiments failed to show NS3h cooperativity in ssDNA binding (20Levin M.K. Patel S.S. J. Biol. Chem. 2002; 277: 29377-29385Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Yet paradoxically it appears that multiple HCV helicase molecules are necessary for efficient DNA unwinding. Classes of helicases show varying levels of intersubunit interactions. For ring helicases, the assembly into a stable cooperative hexamer ring is necessary for activity (1Patel S.S. Picha K.M. Annu. Rev. Biochem. 2000; 69: 651-697Crossref PubMed Scopus (462) Google Scholar, 21Kato M. Frick D.N. Lee J. Tabor S. Richardson C.C. Ellenberger T. J. Biol. Chem. 2001; 276: 21809-21820Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Whether non-ring helicases function as monomers or cooperatively as oligomers is an active area of research. Cooperative behavior in helicases can be manifested as coordination in ATPase, nucleic acid binding, or translocation activities. Such cooperativity arises from physical interaction between the helicase molecules, and it is characterized by a certain free energy of binding. However, a different type of cooperative behavior that does not require protein-protein interactions arises from the mere presence of multiple helicase molecules on the same nucleic acid substrate. The superfamily 1 helicases Rep and UvrD helicases from E. coli have been proposed to function as dimers (22Cheng W. Hsieh J. Brendza K.M. Lohman T.M. J. Mol. Biol. 2001; 310: 327-350Crossref PubMed Scopus (118) Google Scholar, 23Maluf N.K. Fischer C.J. Lohman T.M. J. Mol. Biol. 2003; 325: 913-935Crossref PubMed Scopus (163) Google Scholar), but multiple UvrD helicase molecules on the DNA show increased activity (23Maluf N.K. Fischer C.J. Lohman T.M. J. Mol. Biol. 2003; 325: 913-935Crossref PubMed Scopus (163) Google Scholar, 24Runyon G.T. Wong I. Lohman T.M. Biochemistry. 1993; 32: 602-612Crossref PubMed Scopus (83) Google Scholar, 25Ali J.A. Maluf N.K. Lohman T.M. J. Mol. Biol. 1999; 293: 815-834Crossref PubMed Scopus (93) Google Scholar). RecQ and PcrA helicases have been proposed to function as monomers (26Xu H.Q. Deprez E. Zhang A.H. Tauc P. Ladjimi M.M. Brochon J.C. Auclair C. Xi X.G. J. Biol. Chem. 2003; 278: 34925-34933Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 27Velankar S.S. Soultanas P. Dillingham M.S. Subramanya H.S. Wigley D.B. Cell. 1999; 97: 75-84Abstract Full Text Full Text PDF PubMed Scopus (671) Google Scholar), but it is not known if multiple helicase molecules have an increased activity. Dda helicase functions as a monomer during DNA unwinding (28Nanduri B. Byrd A.K. Eoff R.L. Tackett A.J. Raney K.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14722-14727Crossref PubMed Scopus (81) Google Scholar) but appears to have optimal streptavidin displacement activity as an oligomer (29Morris P.D. Raney K.D. Biochemistry. 1999; 38: 5164-5171Crossref PubMed Scopus (113) Google Scholar). We have characterized the interactions of HCV helicase with DNA substrates of varying ssDNA tail lengths to determine the number of NS3h molecules bound to a helicase substrate. To determine the degree of cooperativity of N53, a superfamily 2 helicase, we measured the unwinding of DNAs containing increasing lengths of the ssDNA tail. We found that multiple helicase molecules are necessary for efficient DNA unwinding. The increase in activity with increasing tail length can be explained by a functional interaction model of unwinding. In this paper we present a way to simulate and fit the helicase unwinding experimental data. We have made this model general by including features that can be applicable to all helicases and used to determine the helicase parameters such as cooperativity in helicase action, processivity of unwinding, stepping rate, and the kinetic step size. The helicase domain of HCV NS3 protein with a C-terminal His tag (NS3h) was expressed in E. coli carrying a plasmid pET21b-NS3HCV (12Kim D.W. Gwack Y. Han J.H. Choe J. Biochem. Biophys. Res. Commun. 1995; 215: 160-166Crossref PubMed Scopus (290) Google Scholar). NS3h protein was purified, stored, and quantitated as described previously (17Levin M.K. Patel S.S. J. Biol. Chem. 1999; 274: 31839-31846Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The reaction buffer contained 50 mm MOPS·NaOH, 5 mm MgCl2, 5 mm dl-dithiothreitol, and 0.1% Tween 20, pH 7.0. Experiments were performed at 22 °C unless specified otherwise. PolyU, average length of 210 nucleotides, was purchased from Amersham Biosciences, and polyoxyethelenesorbitanmonolaurate (Tween 20), purchased from Sigma, was purified by passing through charcoal. DNA Substrates—The sequences of the oligodeoxynucleotides shown in Table I were produced using a random number algorithm and checked for lack of stable secondary structure with the Oligo Analyzer (www.idtdna.com). The oligodeoxynucleotides were synthesized by Integrated DNA Technologies and purified by PAGE in 7 m urea, 60 °C. The concentration of DNA was determined spectrophotometrically in 8 m urea using extinction coefficients of the individual bases. The dsDNA substrates were annealed by heating to 95 °C followed by gradual cooling. The completeness of annealing was checked by native PAGE.Table IDNA substrates Open table in a new tab Electron Microscopy—200 nm NS3h protein was mixed with 50 nm 66 bp-25 DNA substrate (see Table I) and cross-linked with 0.1% glutaraldehyde. The cross-linked complexes were purified on a column packed with A5M gel filtration resin (Bio-Rad) and adsorbed on a glow-charged carbon-coated copper grid. The adsorbed complexes were dehydrated by washing with increasing concentrations of ethanol in water, dried, and rotary shadow-cast with tungsten. The samples were examined and photographed using a Philips 420 electron microscope under 50,000× magnification. Fluorimetric Titration—Fluorimetric titration was performed as described previously (20Levin M.K. Patel S.S. J. Biol. Chem. 2002; 277: 29377-29385Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Briefly, aliquots of DNA solution were added to the solution of NS3h protein and allowed to mix before measuring its absorbance at 280 nm and fluorescence after excitation at 280 nm at an emission wavelength of 340 nm. Values of fluorescence were corrected for dilution and inner filter effect and plotted against [DNA], and the [DNA]-dependent fluorescence change was fit to Equation 1, F=fE×(Et−Eb)+fEb×Eb (Eq. 1) where F is the observed fluorescence, fE is the fluorescence coefficient of free NS3h, fEb is the fluorescence coefficient of NS3h bound to DNA, Et is total NS3h concentration, and Eb is concentration of NS3h bound to DNA, and Equation 2, Eb=Kd+Et+Dt×n−(Kd+Et+Dt×n)2−4×Et×Dt×n2Â(Eq. 2) where Dt is total DNA concentration, Kd is the dissociation constant, and n is the number of NS3h molecules per DNA. Helicase Assay—The kinetics of dsDNA unwinding was measured using RQF-3 Quench-Flow apparatus (KinTek Instruments, Austin, TX). NS3h (0.5 μm) was mixed with 2 nm ss/dsDNA substrate with radiolabeled shorter strand, incubated for 10 s, and mixed with 5 mm ATP and 0.5 μm polyU for 0.1–65 s (concentrations are given for the state after the second mixing). The unwinding reaction was stopped by ejecting the mixture into a test tube containing 100 mm EDTA, 3% SDS, 15% Ficoll, and 0.1% of bromphenol blue. The intact and unwound DNA substrates were resolved on a native 10 or 13% PAGE, and their radioactivity was measured with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The fraction of unwound substrate was calculated and corrected for the presence of ssDNA at time 0 using Equation 3, F=SS×DS0−SS0×DSDS0×(SS+DS) (Eq. 3) where F is the fraction of unwound substrate, DS and SS are radioactivities of intact and unwound substrate bands at a given time, respectively, and DS0 and SS0 are the radioactivities of intact and unwound substrates at time 0, respectively. Data Analysis—The unwinding time courses were fit to the incomplete gamma function (Equation 4), F=A∫0∞e−xxn−1dx∫0kte−xxn−1dx (Eq. 4) where F is a fraction of unwound DNA substrate molecules, A is the amplitude of unwinding, k is stepping rate, and t is reaction time. The number of steps, n, taken by the helicase to unwind the substrate was calculated as n=Lds−Las (Eq. 5) where Lds is the number of base pairs in the DNA substrate duplex, La is the length of the shortest DNA duplex that can stay together under the experimental conditions, and s is the step size. Software MATLAB with Optimization toolbox (The MathWorks, Inc., Natick, MA) was used for all calculations. Electron Microscopy (EM) of NS3h·ss/dsDNA Complexes— EM was used to determine the interactions of NS3h with the ssDNA and duplex DNA regions of the partial duplex helicase substrate. An excess of NS3h was incubated with the 66 bp-25 DNA substrate that has a 25-nt ssDNA tail and a 66-bp duplex (Table I). The complex was immobilized on a carbon grid, tungsten shadow-cast, and visualized by EM. Fig. 1 shows the electron micrograph of the protein-DNA complexes that appear as a ball and a stick. The "stick" part of a complex has a thickness of 0.4 nm, which is typical for dsDNA visualized by tungsten shadow casting. The average length of the stick, 17.3 ± 2 nm, is close to the theoretical length of 66 bp B-DNA (22.4 nm). The ssDNA is not visible by the EM method used; therefore, the "ball" flanking the stick represents ssDNA covered with the NS3h protein. The EM studies show that the complex of multiple NS3h molecules bound to ssDNA is not extended, like e.g. the complex of T7 gp4, RecA, or E. coli single-stranded DNA-binding protein with ssDNA (30Egelman E.H. Yu X. Wild R. Hingorani M.M. Patel S.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3869-3873Crossref PubMed Scopus (253) Google Scholar, 31Egelman E.H. Stasiak A. J. Mol. Biol. 1986; 191: 677-697Crossref PubMed Scopus (189) Google Scholar, 32Chrysogelos S. Griffith J. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 5803-5807Crossref PubMed Scopus (145) Google Scholar). Instead, NS3h-ssDNA complex forms a clump regardless of the length of ssDNA (data not shown). In addition, the micrographs of NS3h-ss·dsDNA complexes show that the NS3h protein binds only to the ssDNA tail of the DNA substrate. Therefore, DNA unwinding does not initiate internally from multiple positions within the dsDNA but must proceed sequentially from the ssDNA tail end. The NS3h Binding to ss/dsDNA Substrates Is Proportional to the ssDNA Tail Length—The stoichiometry and the Kd of the NS3h complex with the ss/ds substrates were measured by fluorimetric titrations. The ss/dsDNA helicase substrates contained a 33-bp duplex region and ssDNA tails ranging in length from 5 to 75 nts (33 bp–5 to 33bp–75 in Table I). The ss/dsDNA helicase substrates were made by annealing synthetic oligodeoxynucleotides. The completeness of annealing was greater than 98% as determined by native PAGE (data not shown). As shown previously, the intrinsic fluorescence of NS3h protein decreases upon binding to ss nucleic acids (33Preugschat F. Averett D.R. Clarke B.E. Porter D.J.T. J. Biol. Chem. 1996; 271: 24449-24457Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). To measure DNA binding, the titration experiments were performed by adding increasing amounts of the ss/dsDNA substrate to a constant amount of NS3h while exciting the sample with 280-nm light and measuring the intrinsic NS3h fluorescence at 340 nm. The titration was repeated with DNA substrates of different ssDNA tail lengths. The resulting fluorescence values were corrected for sample dilution and inner filter effect. To determine the number of NS3h proteins bound per ss/dsDNA substrate (n) and the Kd, the fluorimetric data were fit to Equations 1 and 2. The number of NS3hs per ss/dsDNA substrate as a function of the ssDNA tail length is shown in Fig. 2A. The number of NS3h proteins bound per DNA substrate increases linearly as the ssDNA tail length increases with a slope of 0.162 ± 0.01 NS3h molecules/base. This corresponds to the NS3h occlusion site of 6.2 ± 0.4 bases. This value is smaller than the occlusion site obtained from similar experiments with the ssDNA substrates (7.7 ± 1 bases) (20Levin M.K. Patel S.S. J. Biol. Chem. 2002; 277: 29377-29385Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). As indicated previously (20Levin M.K. Patel S.S. J. Biol. Chem. 2002; 277: 29377-29385Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), the observed binding behavior of NS3h protein with long ssDNA substrates is a product of binding events to multiple overlapping sites. A slightly higher binding density and a smaller occlusion site can result if NS3h has a higher affinity for binding to the ss/dsDNA junction. The Kd for the 33 bp-10 ss/dsDNA substrate with a 10-nt ssDNA tail is 1.3 ± 0.5 nm (Fig. 2B). This value is consistent with Kd values previously reported for short ssDNA substrates (20Levin M.K. Patel S.S. J. Biol. Chem. 2002; 277: 29377-29385Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). As in the case of ssDNA substrates, no cooperativity was detected in the binding of NS3h to the ss/dsDNA substrates, and equations describing a simple non-cooperative binding model fit well to the fluorimetric data (Equations 1 and 2). The DNA Unwinding Efficiency of the NS3h Is Dependent on the Duplex as Well as the ssDNA Tail Length—The unwinding kinetics were measured using a rapid quench-flow instrument as shown in Fig. 3A. The helicase and DNA were preincubated for 10 s, and increasing the preincubation time by 3-fold did not change the unwinding kinetics (data not shown). Thus, a functional helicase-DNA complex is formed within 10 s, and lengthy preincubation times were not necessary to observe activity as reported for NS3 by Pang et al. (10Pang P.S. Jankowsky E. Planet P.J. Pyle A.M. EMBO J. 2002; 21: 1168-1176Crossref PubMed Scopus (205) Google Scholar). Helicase substrates of different duplex lengths from 18-bp to 40-bp and of varying ssDNA tail length, from 5 to 75 nt were studied. Two kinds of substrates with varying ssDNA tails were used. One set of substrates (18-bp and 33-bp) contained tails of a defined sequence shown in Table I and the second set (18-bp, 33-bp, 40-bp) contained dTn tails. After pre-incubating NS3h with the DNA substrate for 10 s, unwinding was initiated by the addition of ATP and polyU RNA. PolyU was used as a trap to prevent NS3h from re-binding to the unwinding substrate once reaction was initiated. Thus, DNA unwinding was measured under single turnover conditions. As shown in Fig. 3, B–F, the substrates with DNA length >18-bp duplex unwound with a kinetic lag, which indicates that the reaction involves a sequence of transient intermediates before the formation of the final product. The single turn-over kinetics of DNA unwinding by UvrD and recBCD helicases have been described by an equation for a multistep process (stepping equation) (34Ali J.A. Lohman T.M. Science. 1997; 275: 377-380Crossref PubMed Scopus (231) Google Scholar, 35Lucius A.L. Maluf N.K. Fischer C.J. Lohman T.M. Biophys. J. 2003; 85: 2224-2239Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). The stepping equation describes the process of dsDNA unwinding as a sequence of identical steps, although the exact nature of these steps has not been determined for any helicase. The same process can be described by the incomplete gamma function (Equation 4), which is continuous with respect to n, the number of steps required to unwind a particular length of duplex. This property makes the incomplete gamma a more convenient function for curve-fitting. We globally fit the unwinding kinetics of DNA with varying ssDNA tails and duplex lengths to a single kinetic step size and stepping rate (Equations 4 and 5). The resulting fit is shown as the solid lines in Fig. 3, B–F. The global fit provided a kinetic step size of 9.1 (±0.9) bp, stepping rate of 0.3 (±0.02) step/s, and a minimal duplex length of 6.6 (±0.9) bp, which is La, the length of the shortest DNA duplex that can stay together under the experimental conditions. Evidently, the number of NS3h molecules bound to the ssDNA tail does not affect the kinetic step size or the rate of unwinding. This indicates that the number of helicase molecules assembled on the ssDNA tail does not change the mechanism by which unwinding occurs. However, the type of helicase complex assembled on the ssDNA does affect the unwinding efficiency (the amount of DNA unwound in a single turnover). As shown in Fig. 4, the unwinding efficiency increases as the ssDNA tail length increases, and shorter duplexes are unwound more efficiently compared with longer duplexes. The length of the ssDNA tail determines the number of NS3h molecules bound to the ss/dsDNA substrate before unwinding begins. The 18-bp duplex was unwound significantly even from a short 5-base ssDNA tail, but neither 5 nor 10 base tails were sufficient to unwind the 33-bp duplex. In the case of the 33- and 40-bp duplex substrates, the efficiency of unwinding increases gradually with the ssDNA tail length, reaching 33 and 20%, respectively, for the 75-base tail. The results also show that the efficiency of unwinding is not affected by the base composition of the ssDNA tail. The amplitudes of unwinding DNA substrates with a tail containing a defined sequence of all bases versus a dTn tail are similar. Although fitting the unwinding data to the incomplete gamma function provided an estimate of the step size and stepping rate, these parameters do not help us to understand the dependence of the unwinding amplitude on the duplex length and the ssDNA tail length. We have used a C-terminal His-tagged helicase domain protein for our studies that show a DNA unwinding rate of 2.7 bp/s at 22 °C (stepping rate × step size). 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 (10Pang P.S. Jankowsky E. Planet P.J. Pyle A.M. EMBO J. 2002; 21: 1168-1176Crossref PubMed Scopus (205) Google Scholar, 11Frick D.N. Rypma R.S. Lam A.M. Gu B. J. Biol. Chem. 2004; 279: 1269-1280Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 36Tackett A.J. Wei L. Cameron C.E. Raney K.D. Nucleic Acids Res. 2001; 29: 565-572Crossref PubMed Scopus (62) Google Scholar). It was also shown recently that the DNA unwinding activity of the helicase domain with C-terminal or N-terminal His tag was similar (11Frick D.N. Rypma R.S. Lam A.M. Gu B. J. Biol. Chem. 2004; 279: 1269-1280Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Therefore, it is unlikely that Hi
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