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• W2000581423 abstract "The structure of the complex of the Escherichia coli primary replicative helicase DnaB protein with single-stranded (ss) DNA and replication fork substrates has been examined using the fluorescence energy transfer method. In these experiments, we used the DnaB protein variant, R14C, which has arginine 14 replaced by cysteine in the small 12-kDa domain of the protein using site-directed mutagenesis. The cysteine residues have been modified with a fluorescent marker which serves as a donor or an acceptor to another fluorescence label placed in different locations on the DNA substrates. Using the multiple fluorescence donor-acceptor approach, we provide evidence that, in the complex with the enzyme, ssDNA passes through the inner channel of the DnaB hexamer. This is the first evidence of the existence of such a structure of a hexameric helicase-ssDNA complex in solution. In the stationary complex with the 5′ arm of the replication fork, without ATP hydrolysis, the distance between the 5′ end of the arm and the 12-kDa domains of the hexamer (R = 47 Å) is the same as in the complex with the isolated ssDNA oligomer (R = 47 Å) having the same length as the arm of the fork. These data indicate that both ssDNA and the 5′ arm of the fork bind in the same manner to the DNA binding site. Moreover, in the complex with the helicase, the length of the ssDNA is similar to the length of the ssDNA strand in the double-stranded DNA conformation. In the stationary complex, the helicase does not invade the duplex part of the fork beyond the first 2–3 base pairs. This result corroborates the quantitative thermodynamic data which showed that the duplex part of the fork does not contribute to the free energy of binding of the enzyme to the fork. Implications of these results for the mechanism of a hexameric helicase binding to DNA are discussed. The structure of the complex of the Escherichia coli primary replicative helicase DnaB protein with single-stranded (ss) DNA and replication fork substrates has been examined using the fluorescence energy transfer method. In these experiments, we used the DnaB protein variant, R14C, which has arginine 14 replaced by cysteine in the small 12-kDa domain of the protein using site-directed mutagenesis. The cysteine residues have been modified with a fluorescent marker which serves as a donor or an acceptor to another fluorescence label placed in different locations on the DNA substrates. Using the multiple fluorescence donor-acceptor approach, we provide evidence that, in the complex with the enzyme, ssDNA passes through the inner channel of the DnaB hexamer. This is the first evidence of the existence of such a structure of a hexameric helicase-ssDNA complex in solution. In the stationary complex with the 5′ arm of the replication fork, without ATP hydrolysis, the distance between the 5′ end of the arm and the 12-kDa domains of the hexamer (R = 47 Å) is the same as in the complex with the isolated ssDNA oligomer (R = 47 Å) having the same length as the arm of the fork. These data indicate that both ssDNA and the 5′ arm of the fork bind in the same manner to the DNA binding site. Moreover, in the complex with the helicase, the length of the ssDNA is similar to the length of the ssDNA strand in the double-stranded DNA conformation. In the stationary complex, the helicase does not invade the duplex part of the fork beyond the first 2–3 base pairs. This result corroborates the quantitative thermodynamic data which showed that the duplex part of the fork does not contribute to the free energy of binding of the enzyme to the fork. Implications of these results for the mechanism of a hexameric helicase binding to DNA are discussed. The DnaB protein is the Escherichia coli primary replicative helicase, i.e. the factor responsible for unwinding the duplex DNA in front of the replication fork (1LeBowitz J.H. McMacken R. J. Biol. Chem. 1986; 261: 4738-4748Abstract Full Text PDF PubMed Google Scholar, 2Baker T.A. Funnell B.E. Kornberg A. J. Biol. Chem. 1987; 262: 6877-6885Abstract Full Text PDF PubMed Google Scholar, 3West S.C. Cell. 1996; 86: 177-180Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). The enzyme is an essential replication protein in E. coli (4Kornberg A. Baker T.A. DNA Replication. Freeman, San Francisco1992Google Scholar) which is involved in both the initiation and elongation stages of DNA replication (3West S.C. Cell. 1996; 86: 177-180Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 5Wickner S. Wright M. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1973; 71: 783-787Crossref Scopus (59) Google Scholar, 6McMacken R. Kornberg A. J. Biol. Chem. 1978; 253: 3313-3319Abstract Full Text PDF PubMed Google Scholar). The DnaB helicase is the only helicase required to reconstitute DNA replication in vitro from the chromosomal origin of replication. In the complex with ssDNA, 1The abbreviations used are: ss, single-stranded; ds, double-stranded; AMP-PNP, β,γ-imidoadenosine-5′-triphosphate; EM, electron microscopy; CPM, 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin; Fl, fluorescein; Rh, rhodamine. the DnaB protein forms a “mobile replication promoter.” This nucleoprotein complex plays an activating role for the primase in the initial stages of the priming reaction (4Kornberg A. Baker T.A. DNA Replication. Freeman, San Francisco1992Google Scholar). Sedimentation equilibrium, sedimentation velocity, and nucleotide cofactor binding studies show that the DnaB helicase exists as a stable hexamer in a large protein concentration range stabilized specifically by magnesium cations (7Bujalowski W. Klonowska M.M. Jezewska M.J. J. Biol. Chem. 1994; 269: 31350-31358Abstract Full Text PDF PubMed Google Scholar, 8Jezewska M.J. Bujalowski W. J. Biol. Chem. 1996; 271: 4261-4265Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 9Jezewska M.J. Kim U.-S. Bujalowski W. Biochemistry. 1996; 35: 2129-2145Crossref PubMed Scopus (83) Google Scholar). Hydrodynamic and EM data indicate that six protomers aggregate with cyclic symmetry in which the protomer-protomer contacts are limited to only two neighboring subunits (7Bujalowski W. Klonowska M.M. Jezewska M.J. J. Biol. Chem. 1994; 269: 31350-31358Abstract Full Text PDF PubMed Google Scholar, 10San Martin M.C. Valpuesta J.M. Stamford N.P.J. Dixon N.E. Carazo J.M. J. Struct. Biol. 1995; 114: 167-176Crossref PubMed Scopus (131) Google Scholar, 11Yu X. Jezewska M.J. Bujalowski W. Egelman E.H. J. Mol. Biol. 1996; 259: 7-14Crossref PubMed Scopus (133) Google Scholar). Hydrodynamic and EM studies also provide direct evidence of the presence of long range allosteric interactions in the hexamer, encompassing all six subunits of the enzyme (7Bujalowski W. Klonowska M.M. Jezewska M.J. J. Biol. Chem. 1994; 269: 31350-31358Abstract Full Text PDF PubMed Google Scholar, 11Yu X. Jezewska M.J. Bujalowski W. Egelman E.H. J. Mol. Biol. 1996; 259: 7-14Crossref PubMed Scopus (133) Google Scholar, 12Bujalowski W. Jezewska M.J. Biochemistry. 1995; 34: 8513-8519Crossref PubMed Scopus (121) Google Scholar). Recently, we obtained the first estimate of the stoichiometry of the DnaB helicase-ssDNA complex and the mechanism of the binding (12Bujalowski W. Jezewska M.J. Biochemistry. 1995; 34: 8513-8519Crossref PubMed Scopus (121) Google Scholar, 13Jezewska M.J. Bujalowski W. Biochemistry. 1996; 35: 2117-2128Crossref PubMed Scopus (84) Google Scholar). In the complex with ssDNA, the DnaB helicase binds the nucleic acid with a stoichiometry of 20 ± 3 nucleotides per DnaB hexamer, and this stoichiometry is independent of the type of nucleic acid base (9Jezewska M.J. Kim U.-S. Bujalowski W. Biochemistry. 1996; 35: 2129-2145Crossref PubMed Scopus (83) Google Scholar,13Jezewska M.J. Bujalowski W. Biochemistry. 1996; 35: 2117-2128Crossref PubMed Scopus (84) Google Scholar). Our thermodynamic studies of binding of the DnaB hexamer to different ssDNA oligomers show that the enzyme has a single, strong binding site for ssDNA. Moreover, the same binding site is used in the binding to oligomers, polymer DNA, and replication fork substrates (9Jezewska M.J. Kim U.-S. Bujalowski W. Biochemistry. 1996; 35: 2129-2145Crossref PubMed Scopus (83) Google Scholar,12Bujalowski W. Jezewska M.J. Biochemistry. 1995; 34: 8513-8519Crossref PubMed Scopus (121) Google Scholar, 13Jezewska M.J. Bujalowski W. Biochemistry. 1996; 35: 2117-2128Crossref PubMed Scopus (84) Google Scholar, 14Jezewska M.J. Rajendran S. Bujalowski W. Biochemistry. 1997; 36: 10320-10326Crossref PubMed Scopus (47) Google Scholar). Photo-cross-linking experiments indicate that the ssDNA binding site is located predominately, if not completely, on a single subunit of the hexamer (9Jezewska M.J. Kim U.-S. Bujalowski W. Biochemistry. 1996; 35: 2129-2145Crossref PubMed Scopus (83) Google Scholar, 12Bujalowski W. Jezewska M.J. Biochemistry. 1995; 34: 8513-8519Crossref PubMed Scopus (121) Google Scholar, 13Jezewska M.J. Bujalowski W. Biochemistry. 1996; 35: 2117-2128Crossref PubMed Scopus (84) Google Scholar). Our data show that, in the complex with the replication fork DNA substrates, the DnaB helicase preferentially binds to the 5′ arm of the fork (14Jezewska M.J. Rajendran S. Bujalowski W. Biochemistry. 1997; 36: 10320-10326Crossref PubMed Scopus (47) Google Scholar). The 3′ arm does not form a stable complex with the DnaB hexamer associated with the 5′ arm, and the 3′ arm is in a conformation in which it is accessible for the binding of another DnaB hexamer. Moreover, the duplex part of the fork substrate does not significantly contribute to the free energy of binding which predominantly comes from interactions with the 5′ arm (14Jezewska M.J. Rajendran S. Bujalowski W. Biochemistry. 1997; 36: 10320-10326Crossref PubMed Scopus (47) Google Scholar). Formulating a physical model of a hexameric helicase mechanism requires the knowledge of the structure of the helicase-ssDNA complex. In phage T7 helicase/primase and E. coli RuvB protein systems, EM data indicated that, in the complex with the enzymes, the ssDNA passes through the inner channel of the protein hexamer (15Stasiak A. Tsaneva I.R. West S.C. Benson C.J.B. Yu X. Egelman E.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7618-7622Crossref PubMed Scopus (143) Google Scholar, 16Egelman 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). On the other hand, an outside mode of ss nucleic acid binding has been proposed for the hexamer of the SV40 T large antigen helicase (17SenGupta D.J. Borowiec J.A. Science. 1992; 256: 1656-1661Crossref PubMed Scopus (58) Google Scholar). In this communication, the structure of the DnaB helicase-ssDNA complex has been studied using the fluorescence energy transfer method. We present evidence that, in the complex with the DnaB hexamer, the ssDNA oligomer, which occupies the entire total DNA binding site of the DnaB helicase, passes through the inner channel of the hexamer. The results indicate that in the stationary complex with the replication fork substrate, the helicase does not invade the duplex part of the fork beyond the first 2–3 base pairs. All solutions were made with distilled and deionized >18 megohms (Milli-Q Plus) water. All chemicals were reagent grade. Buffer T2 is 50 mm Tris adjusted to pH 8.1 with HCl, 5 mm MgCl2, and 10% glycerol. The temperatures and concentrations of NaCl and AMP-PNP in the buffer are indicated in the text. The E. coli DnaB protein was purified, as we described previously (18Bujalowski W. Klonowska M.M. Biochemistry. 1993; 32: 5888-5900Crossref PubMed Scopus (104) Google Scholar, 19Bujalowski W. Klonowska M.M. Biochemistry. 1994; 33: 4682-4694Crossref PubMed Scopus (55) Google Scholar). The concentration of the protein was spectrophotometrically determined using the extinction coefficient, ε280 = 1.85 × 105 cm−1m−1 (hexamer) (7Bujalowski W. Klonowska M.M. Jezewska M.J. J. Biol. Chem. 1994; 269: 31350-31358Abstract Full Text PDF PubMed Google Scholar). All nucleic acids were purchased from Midland Certified Reagents (Midland, TX). The 20 mer dT(pT)19, labeled with fluorescein at the 5′ end, or at a different location of the nucleic acid, were synthesized using fluorescein phosphoramidate (Glen Research). Labeling of 20 mers at the 3′ end with fluorescein, or labeling with rhodamine (Rh), was performed by synthesizing dT(pT)19 with a nucleotide residue in a given location of the nucleic acid with the amino group on a six-carbon linker and, subsequently, modifying the amino group with fluorescein 5′-isothiocyanate or tetramethylrhodamine 6-isothiocyanate (Midland Certified Reagents). The degree of labeling was determined by absorbance at 494 nm for fluorescein (pH 9) using the extinction coefficient, ε494 = 7.6 × 104m−1 cm−1, and at 555 nm for rhodamine using the extinction coefficient, ε555 = 8.0 × 104m−1 cm−1 (9Jezewska M.J. Kim U.-S. Bujalowski W. Biochemistry. 1996; 35: 2129-2145Crossref PubMed Scopus (83) Google Scholar). Concentrations of all ssDNA oligomers have been spectrophotometrically determined, using the nearest-neighbor analysis (9Jezewska M.J. Kim U.-S. Bujalowski W. Biochemistry. 1996; 35: 2129-2145Crossref PubMed Scopus (83) Google Scholar, 20Cantor C.R. Warshaw M.M. Shapiro H. Biopolymers. 1970; 9: 1059-1077Crossref PubMed Scopus (879) Google Scholar). The single-arm fork substrates were obtained by mixing the proper oligomers at given concentrations, warming up the mixture for 5 min at 95 °C, and slowly cooling for a period of ∼2 h (14Jezewska M.J. Rajendran S. Bujalowski W. Biochemistry. 1997; 36: 10320-10326Crossref PubMed Scopus (47) Google Scholar). Replacement of the arginine residues at position 14 from the N terminus of the DnaB protein and obtaining the DnaB protein variant, R14C, were performed using the plasmid RLM1038, harboring the gene of the wild type DnaB helicase. 2Jezewska, M. J., Rajendran, S., and Bujalowski, W. (1998) J. Biol. Chem. 273, 9058–9069 The site-directed mutagenesis was accomplished in the NIEHS Center facility directed by Dr. T. Wood. Labeling of the six cysteine residues of the DnaB variant, R14C hexamer, with CPM or fluorescein 5-maleimide was performed in H buffer (50 mm Hepes/HCl, pH 8.1, 100 mm NaCl, 5 mm MgCl2, 10% glycerol) at 4 °C. The fluorescent label was added from the stock solution to the molar ratio of the dye/R14C ≈ 25. The mixture was incubated for 4 h, with gentle mixing. After incubation, the protein was precipitated with ammonium sulfate and dialyzed overnight against buffer T2. Any remaining free dye was removed from the modified R14C-CPM or R14C-Fl by applying the sample on a DEAE-cellulose column and eluting with buffer T2 containing 500 mm NaCl. The degree of labeling, γ, was determined by the absorbance of a marker using the extinction coefficient, ε394 = 27 × 103 cm−1m−1 for CPM and ε494 = 78 × 103 cm−1m−1 for fluorescein, respectively. The obtained values of γ were 5.8 ± 0.1 for CPM and 5.7 ± 0.1 for fluorescein, indicating that all cysteine residues in R14C are readily available for modification. 3S. Rajendran, M. J. Jezewska, and W. Bujalowski, manuscript in preparation. All steady-state fluorescence titrations were performed using the SLM-AMINCO 48000S and 8100 spectrofluorometers (12Bujalowski W. Jezewska M.J. Biochemistry. 1995; 34: 8513-8519Crossref PubMed Scopus (121) Google Scholar, 21Jezewska M.J. Bujalowski W. Biophys. Chem. 1997; 64: 253-269Crossref PubMed Scopus (37) Google Scholar, 22Jezewska M.J. Kim U-S. Bujalowski W. Biophys. J. 1996; 71: 2075-2086Abstract Full Text PDF PubMed Scopus (41) Google Scholar). The emission spectra have been corrected for instrument characteristics using the software provided by the manufacturer. Fluorescence anisotropy measurements were performed in the L format, using Glan-Thompson polarizers placed in the excitation and emission channels. The fluorescence anisotropy of the sample was calculated using (23Lakowicz J.R. Principle of Fluorescence Spectroscopy. Plenum Press, New York1983Crossref Google Scholar) r=(IVV−GIVH)(IVV+2GIVH)Equation 1 were I is the fluorescence intensity and the first and the second subscripts refer to vertical (V) polarization of the excitation and vertical (V) or horizontal (H) polarization of the emitted light (19Bujalowski W. Klonowska M.M. Biochemistry. 1994; 33: 4682-4694Crossref PubMed Scopus (55) Google Scholar). The factor G = I HV/I HH corrects for the different sensitivity of the emission monochromator for vertically and horizontally polarized light (24Azumi T. McGlynn S.P. J. Chem. Phys. 1962; 37: 2413-3240Crossref Scopus (423) Google Scholar). The limiting fluorescence anisotropies of fluorophores, r lim, were determined by measuring the anisotropy of a given sample at a different solution viscosity, adjusted by sucrose or glycerol, and extrapolating to viscosity = ∞, using the Perrin equation (19Bujalowski W. Klonowska M.M. Biochemistry. 1994; 33: 4682-4694Crossref PubMed Scopus (55) Google Scholar,23Lakowicz J.R. Principle of Fluorescence Spectroscopy. Plenum Press, New York1983Crossref Google Scholar, 25Bujalowski W. Klonowska M.M. J. Biol. Chem. 1994; 269: 31359-31371Abstract Full Text PDF PubMed Google Scholar). The efficiency of the fluorescence radiationless energy transfer, E, from donors located on the small 12-kDa domains of the DnaB protein variant R14C, to an acceptor located on a DNA substrate bound in the DNA binding site of the DnaB helicase, has been determined using two independent methods. The fluorescence of the donor in the presence of the acceptor, F DA, is related to the fluorescence of the same donor, F D, in the absence of the acceptor by FDA=(1−νD)FD+FDνD(1−ED)Equation 2a where ν D is the fraction of donors in the complex with the acceptor, and E D is the average fluorescence energy transfer from a donor to an acceptor, determined from the quenching of the donor fluorescence. Thus, the average transfer efficiency, E D, obtained from the quenching of the donor fluorescence is obtained by rearranging Equation 2a ED=1νDFD−FDAFDEquation 2b The values of ν D have been determined using the binding constants of a given DNA substrate for the DnaB helicase measured in the same solution conditions (9Jezewska M.J. Kim U.-S. Bujalowski W. Biochemistry. 1996; 35: 2129-2145Crossref PubMed Scopus (83) Google Scholar, 14Jezewska M.J. Rajendran S. Bujalowski W. Biochemistry. 1997; 36: 10320-10326Crossref PubMed Scopus (47) Google Scholar). In the second independent method, the average fluorescence transfer efficiency, E A, has been determined, using a sensitized acceptor fluorescence, by measuring the fluorescence intensity of the acceptor (fluorescein or rhodamine), excited at a wavelength where a donor predominantly absorbs, in the absence and presence of the donor. The fluorescence intensities of the acceptor in the absence, F A, and presence,F AD, of the donor are defined as FA=IoεACATφFAEquation 3a and FAD=(1−νA)FA+IoεAνACATφBA+IoεDCDTνDφBAEAEquation 3b where I o is the intensity of incident light,C AT and C DT are the total concentrations of acceptor and donor, ν A is the fraction of acceptors in the complex with donors, ε A and ε D are the molar absorption coefficients of acceptor and donor at the excitation wavelength, respectively, φFA and φBA are the quantum yields of the free and bound acceptor, and E A is the average transfer efficiency determined by the acceptor sensitized emission. All quantities in Equations 3a and 3b can be experimentally determined. For the case considered in this work, the acceptor is practically, completely saturated with the donor, i.e. ν A = 1. Thus, for ν A = 1, dividing Equation 3b by 3a and rearranging provides the average transfer efficiency as described by EA=1νDεACATεDCDTφFAφBAFADFA−1Equation 3c It should be pointed out that the energy transfer efficiencies,E D and E A, are apparent quantities. E D is a fraction of the photons absent in the donor emission as a result of the presence of an acceptor, including transfer to the acceptor and possible nondipolar quenching processes induced by the presence of the acceptor, and E A is a fraction of all photons absorbed by the donor which were transferred to the acceptor. The true Förster energy transfer efficiency, E, is a fraction of the photons absorbed by the donor and transferred to the acceptor, in the absence of any additional nondipolar quenching resulting from the presence of the acceptor (23Lakowicz J.R. Principle of Fluorescence Spectroscopy. Plenum Press, New York1983Crossref Google Scholar). The value of E is related to the apparent quantities of E D and E A by (26Berman H.A Yguerabide J. Taylor P. Biochemistry. 1980; 19: 2226-2235Crossref PubMed Scopus (61) Google Scholar) E=EA(1−ED+EA)Equation 4 Thus, measurements of the transfer efficiency, using both methods, are not alternatives but parts of the entire analysis used to obtain the true efficiency of the fluorescence energy transfer process,E. The fluorescence energy transfer efficiency between the donor and acceptor dipoles is related to the distance, R, separating the dipoles by Equations 5a and 5b (23Lakowicz J.R. Principle of Fluorescence Spectroscopy. Plenum Press, New York1983Crossref Google Scholar) R=Ro(1−E)E1/6Equation 5a and Ro=9790(κ2n−4φdJ)1/6Equation 5b where R o is the so-called Förster critical distance (in angstroms), the distance at which the transfer efficiency is 50%, κ2 is the orientation factor, φ d is the donor quantum yield in the absence of the acceptor, and n is the refractive index of the medium (n = 1.4) (23Lakowicz J.R. Principle of Fluorescence Spectroscopy. Plenum Press, New York1983Crossref Google Scholar). The overlap integral, J, characterizes the resonance between the donor and acceptor dipoles and has been evaluated by integration of the mutual area of overlap between the donor emission spectrum, F(λ), and the acceptor absorption spectrum, ε A(λ), as defined by (23Lakowicz J.R. Principle of Fluorescence Spectroscopy. Plenum Press, New York1983Crossref Google Scholar) J=∫F(λ)εA(λ)λ4∂λ∫F(λ)∂λEquation 5c The fluorescence transfer efficiency determined for chemically identical donor-acceptor pairs, characterized by the same donor quantum yields, depends on the distance between the donor and the acceptor,R, and factor κ2, describing the mutual orientation of the donor and acceptor dipoles (23Lakowicz J.R. Principle of Fluorescence Spectroscopy. Plenum Press, New York1983Crossref Google Scholar). The factor κ2 cannot be experimentally determined, however, the upper, κmax2, and the lower, κmin2, limit of κ2 can be estimated from the measured limiting anisotropies of the donor and acceptor and the calculated axial depolarization factors, using the procedure described by Dale et al. (27Dale R.E. Esinger J. Blumberg W.E. Biophys. J. 1979; 26: 161-194Abstract Full Text PDF PubMed Scopus (657) Google Scholar). When both axial depolarization factors are positive, κmax2 and κmin2 can be calculated from κmax2=23(1+〈dDX〉+〈dAX〉+3〈dDX〉〈dAX〉)Equation 6a and κmin2=231−12(〈dDX〉+〈dAX〉)Equation 6b where <dDx> and <dAx> are the axial depolarization factors for the donor and acceptor, respectively (27Dale R.E. Esinger J. Blumberg W.E. Biophys. J. 1979; 26: 161-194Abstract Full Text PDF PubMed Scopus (657) Google Scholar). The axial depolarization factors have been calculated as square roots of the ratios of the limiting anisotropies of the donors and acceptors and their corresponding fundamental anisotropies, r o(r o = 0.4 for CPM and fluorescein) (19Bujalowski W. Klonowska M.M. Biochemistry. 1994; 33: 4682-4694Crossref PubMed Scopus (55) Google Scholar, 25Bujalowski W. Klonowska M.M. J. Biol. Chem. 1994; 269: 31359-31371Abstract Full Text PDF PubMed Google Scholar, 27Dale R.E. Esinger J. Blumberg W.E. Biophys. J. 1979; 26: 161-194Abstract Full Text PDF PubMed Scopus (657) Google Scholar). The parameter κ2 can assume a value from 0 to 4. For complete random orientation of the acceptor and donor, κ2= 0.67 (23Lakowicz J.R. Principle of Fluorescence Spectroscopy. Plenum Press, New York1983Crossref Google Scholar). However, because the distance between a donor and an acceptor depends upon the 1/6th power of κ2, only the two extreme values (0 or 4) would significantly affect the determined distance. It should be pointed out that the analysis of the possible range of distances between the donor and the acceptor, using κmin2 and κmax2, describes the situation where only a single donor-acceptor pair is used. Another equally rigorous procedure to evaluate the error in the distance determination, although more time consuming and much more expensive, is to use multiple donor-acceptor pairs (28Cantor C.R. Pechukas P. Proc. Nat. Acad. Sci. U. S. A. 1971; 68: 2099-2101Crossref PubMed Google Scholar). The different molecular structures of the different donor and acceptors introduce intrinsic randomization of the orientation of the absorption and emission dipoles. The measurement of a very similar distance using a different donor-acceptor pair, indicates that the orientation of the donor and absorption dipoles is far from the extreme values of 0 or 4, and that the true distance between a donor and an acceptor is very close to the distance obtained using κ2 = 0.67. In the system studied in this work, six fluorescent labels, arranged in a ring, are located at one end of the DnaB hexamer (see below). A fluorescence energy homo-transfer between the same fluorophore molecules may occur in such a system which would complicate the molecular distance estimates. The extent of the homo-transfer was examined by determining the fluorescence anisotropy of the DnaB hexamer as a function of the degree of labeling and by excitation fluorescence anisotropy spectra of the fully labeled DnaB hexamer. None of these approaches showed a measurable homo-transfer, even in the case of the R14C-Fl labeled fluorescein which has a large overlap of its absorption and fluorescence spectra (36Weber G. Trans. Faraday Soc. 1954; 50: 552-555Crossref Scopus (108) Google Scholar). If a set of m identical donors transfers the energy to a single acceptor, as in the cases studied in this work, the average transfer efficiency is weighted by the contributions,E i, from all donors and is defined in general as E=1m∑EiEquation 7 where E i is the transfer efficiency from the individual donor, i, to the acceptor. It should be noted that, if all individual transfer efficiencies, E i, are equal, e.g. in the case where the donors are located at the same distance from the acceptor, the experimentally determined average fluorescence transfer efficiency is then E ≈ (1/m)mE i = E i. The quantum yields of different chromophores used in this work, φ, were determined by the comparative method (29Parker C.A. Reese W.T. Analyst. 1960; 85: 587-592Crossref Google Scholar) we previously described (19Bujalowski W. Klonowska M.M. Biochemistry. 1994; 33: 4682-4694Crossref PubMed Scopus (55) Google Scholar). Quinine bisulfate in 0.1 NH2SO4 and fluorescein in 0.1 NaOH were used as a standard (absolute quantum yield φ = 0.7 and 0.92, respectively) (30Scott T.G. Spencer R.D. Leonard N.J. Weber G. J. Am. Chem. Soc. 1970; 92: 687-695Crossref Scopus (361) Google Scholar, 31Weber G. Biochem. J. 1960; 75: 335-345Crossref PubMed Scopus (300) Google Scholar). The DnaB monomer, which has an elongated shape, is built of two structural domains (7Bujalowski W. Klonowska M.M. Jezewska M.J. J. Biol. Chem. 1994; 269: 31350-31358Abstract Full Text PDF PubMed Google Scholar, 10San Martin M.C. Valpuesta J.M. Stamford N.P.J. Dixon N.E. Carazo J.M. J. Struct. Biol. 1995; 114: 167-176Crossref PubMed Scopus (131) Google Scholar, 32Nakayama N. Arai N. Bond M.N. Kaziro Y. Arai K. J. Biol. Chem. 1984; 259: 97-101Abstract Full Text PDF PubMed Google Scholar). A small 12-kDa domain at the N terminus of the protein and a large 33-kDa domain at the C terminus are both connected at the hinge region. This structure of a monomer has been visualized in EM studies which also showed that, in the cyclic DnaB hexamer, all protomers are oriented with the small 12-kDa domain in the same direction (10San Martin M.C. Valpuesta J.M. Stamford N.P.J. Dixon N.E. Carazo J.M. J. Struct. Biol. 1995; 114: 167-176Crossref PubMed Scopus (131) Google Scholar). In R14C-CPM or R14C-Fl, each of the six small 12-kDa domains of the hexamer is labeled with a fluorescent marker, coumarin (CPM) or fluorescein at a specific site (see “Experimental Procedures”). Thus, all six fluorophores in the labeled R14C variant are at the same end of the DnaB hexamer and arranged in a ring. The schematic representation of the DnaB hexamer based on hydrodynamic and EM data is shown in Fig. 1 a. Fluorescence energy transfer from a donor to an acceptor is one of the most intensively used methods in studying macromolecular distances in solution (23Lakowicz J.R. Principle of Fluorescence Spectroscopy. Plenum Press, New York1983Crossref Google Scholar). The overlap of an absorption spectrum of an acceptor with the emission spectrum of a donor is a condition for the fluorescence resonance energy transfer to occur. The fluorescence emission spectrum of R14C-CPM (λex = 425 nm) together with the absorption spectrum of 5′-Fl-dT(pT)19 and 5′-Rh-dT(pT)19, as well as the fluorescence emission spectrum of R14C-Fl (λex = 485 nm), with the absorption spectrum of 5′-Rh-dT(pT)19 in buffer T2 (pH 8.1, 20 °C), containing 100 mm NaCl and 1 mm AMP-PNP, are shown in Fig. 2, a–c. In the case of all three donor-acceptor pairs, there is a very significant spectral overlap of the donor emission with the acceptor absorption spectrum, indicating that efficient fluorescence energy transfer can occur, if the donor and acceptor are in close proximity. F" @default.
• W2000581423 created "2016-06-24" @default.
• W2000581423 creator A5000080769 @default.
• W2000581423 creator A5025276977 @default.
• W2000581423 creator A5032509528 @default.
• W2000581423 creator A5073159818 @default.
• W2000581423 date "1998-04-01" @default.
• W2000581423 modified "2023-09-30" @default.
• W2000581423 title "Does Single-stranded DNA Pass through the Inner Channel of the Protein Hexamer in the Complex with the Escherichia coli DnaB Helicase?" @default.
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