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• W2003660437 abstract "The structure-function relationship within the DNA binding site of the Escherichia coli replicative helicase DnaB protein was studied using nuclease digestion, quantitative fluorescence titration, centrifugation, and fluorescence energy transfer techniques. Nuclease digestion of the enzyme-single-stranded DNA (ssDNA) complexes reveals large structural heterogeneity within the binding site. The total site is built of two subsites differing in structure and affinity, although both occlude ∼10 nucleotides. ssDNA affinity for the strong subsite is ∼3 orders of magnitude higher than that for the weak subsite.Fluorescence energy transfer experiments provide direct proof that the DnaB hexamer binds ssDNA in a single orientation, with respect to the polarity of the sugar-phosphate backbone. This is the first evidence of directional binding to ssDNA of a hexameric helicase in solution. The strong binding subsite is close to the small 12-kDa domains of the DnaB hexamer and occludes the 5′-end of the ssDNA. The strict orientation of the helicase on ssDNA indicates that, when the enzyme approaches the replication fork, it faces double-stranded DNA with its weak subsite. The data indicate that the different binding subsites are located sequentially, with the weak binding subsite constituting the entry site for double-stranded DNA of the replication fork. The structure-function relationship within the DNA binding site of the Escherichia coli replicative helicase DnaB protein was studied using nuclease digestion, quantitative fluorescence titration, centrifugation, and fluorescence energy transfer techniques. Nuclease digestion of the enzyme-single-stranded DNA (ssDNA) complexes reveals large structural heterogeneity within the binding site. The total site is built of two subsites differing in structure and affinity, although both occlude ∼10 nucleotides. ssDNA affinity for the strong subsite is ∼3 orders of magnitude higher than that for the weak subsite. Fluorescence energy transfer experiments provide direct proof that the DnaB hexamer binds ssDNA in a single orientation, with respect to the polarity of the sugar-phosphate backbone. This is the first evidence of directional binding to ssDNA of a hexameric helicase in solution. The strong binding subsite is close to the small 12-kDa domains of the DnaB hexamer and occludes the 5′-end of the ssDNA. The strict orientation of the helicase on ssDNA indicates that, when the enzyme approaches the replication fork, it faces double-stranded DNA with its weak subsite. The data indicate that the different binding subsites are located sequentially, with the weak binding subsite constituting the entry site for double-stranded DNA of the replication fork. The DnaB protein is an essential replication protein inEscherichia coli (1Kornberg A. Baker T.A. DNA Replication. W. H. Freeman and Co., San Francisco1992Google Scholar) which is involved in both the initiation and elongation stages of DNA replication (2Wickner S. Wright M. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1973; 71: 783-787Crossref Scopus (59) Google Scholar, 3McMacken R. Kornberg A. J. Biol. Chem. 1977; 253: 3313-3319Abstract Full Text PDF Google Scholar, 4Ueda K. McMacken R. Kornberg A. J. Biol. Chem. 1978; 253: 261-269Abstract Full Text PDF PubMed Google Scholar). The protein is the E. coli primary replicative helicase, i.e.the factor responsible for unwinding the duplex DNA in front of the replication fork (5LeBowitz J.H. McMacken R. J. Biol. Chem. 1986; 261: 4738-4748Abstract Full Text PDF PubMed Google Scholar, 6Baker T.A. Funnell B.E. Kornberg A. J. Biol. Chem. 1987; 262: 6877-6885Abstract Full Text PDF PubMed Google Scholar). The DnaB protein 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: ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; AMP-PNP, β,γ-imidoadenosine-5′-triphosphate; CPM, 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin; Fl, fluorescein. the DnaB protein forms a “mobile replication promoter.” This nucleoprotein complex is specifically recognized by the primase in the initial stages of the priming reaction (1Kornberg A. Baker T.A. DNA Replication. W. H. Freeman and Co., San Francisco1992Google Scholar). In solution, the native DnaB protein exists as a stable hexamer, composed of six identical subunits (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, 9Reha-Krantz L.J. Hurwitz J. J. Biol. Chem. 1978; 253: 4051-4057Abstract Full Text PDF PubMed Google 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, specifically stabilized 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). Hydrodynamic and electron microscopy 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 (132) Google Scholar). Sedimentation velocity and electron microscopy studies reveal that the DnaB hexamer undergoes dramatic conformational changes upon binding AMP-PNP and ssDNA, and provide direct evidence of the presence of long range allosteric interactions in the hexamer, encompassing all six subunits of the enzyme (8Jezewska M.J. Bujalowski W. J. Biol. Chem. 1996; 271: 4261-4265Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 11Yu X. Jezewska M.J. Bujalowski W. Egelman E.H. J. Mol. Biol. 1996; 259: 7-14Crossref PubMed Scopus (132) 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 (120) Google Scholar, 13Jezewska M.J. Kim U-S. Bujalowski W. Biochemistry. 1996; 35: 2129-2145Crossref PubMed Scopus (82) Google Scholar, 14Jezewska M.J. Kim U-S. Bujalowski W. Biophys. J. 1996; 71: 2075-2086Abstract Full Text PDF PubMed Scopus (41) Google Scholar). Using the quantitative fluorescence titration method, we determined that the DnaB helicase binds ssDNA with a stoichiometry of 20 ± 3 nucleotides/DnaB hexamer and that this stoichiometry is independent of the type of nucleic acid base (13Jezewska M.J. Kim U-S. Bujalowski W. Biochemistry. 1996; 35: 2129-2145Crossref PubMed Scopus (82) Google Scholar). Our thermodynamic studies of binding of ssDNA oligomers to the DnaB hexamer show that the enzyme has a single, strong binding site for ssDNA (12Bujalowski W. Jezewska M.J. Biochemistry. 1995; 34: 8513-8519Crossref PubMed Scopus (120) Google Scholar). The results also show that the same binding site is used in the binding to oligomers and polymer nucleic acids (12Bujalowski W. Jezewska M.J. Biochemistry. 1995; 34: 8513-8519Crossref PubMed Scopus (120) Google Scholar, 13Jezewska M.J. Kim U-S. Bujalowski W. Biochemistry. 1996; 35: 2129-2145Crossref PubMed Scopus (82) Google Scholar). Moreover, photo-cross-linking experiments indicate that the ssDNA binding site is located predominately, if not completely, on a single subunit of the hexamer (12Bujalowski W. Jezewska M.J. Biochemistry. 1995; 34: 8513-8519Crossref PubMed Scopus (120) Google Scholar, 13Jezewska M.J. Kim U-S. Bujalowski W. Biochemistry. 1996; 35: 2129-2145Crossref PubMed Scopus (82) Google Scholar). The reaction catalyzed by a helicase, the unwinding of a duplex DNA, must take place in the DNA binding site. The fact that the helicase uses the same single DNA binding site, when forming a complex with polymer ssDNAs, oligomers, and replication fork substrates, indicates a complex structure of the nucleic acid binding site that can accommodate both ssDNA and dsDNA. In this communication, we report the analysis of interactions between the DnaB helicase and DNA within the total DNA binding site of the enzyme. We present direct evidence that the total DNA binding site of the helicase is structurally and functionally heterogeneous. The total binding site is built of two subsites, each encompassing approximately 10 nucleotide residues. We provide direct proof that the DnaB hexamer binds ssDNA in a strictly single orientation, with respect to the polarity of the sugar-phosphate backbone of the nucleic acid. The results indicate that the binding subsites are sequentially located along the nucleic acid lattice, with the weak binding subsite constituting an entry site for the duplex part of the replication fork. All solutions were made with distilled and deionized >18 megaohms (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, 10% glycerol. Buffer H is 50 mm Hepes adjusted to pH 8.1 with HCl, 5 mm MgCl2, 10% glycerol. The temperature, AMP-PNP, and salt concentrations are indicated in the text. The fluorescent markers, CPM, and fluorescein 5′-isothiocyanate, used in the modification, were purchased from Molecular Probes (Eugene, OR). The E. coli DnaB protein was purified, as described previously by us (7Bujalowski W. Klonowska M.M. Jezewska M.J. J. Biol. Chem. 1994; 269: 31350-31358Abstract Full Text PDF PubMed Google Scholar, 15Bujalowski W. Klonowska M.M. Biochemistry. 1993; 32: 5888-5900Crossref PubMed Scopus (104) Google Scholar, 16Bujalowski W. Klonowska M.M. Biochemistry. 1994; 33: 4682-4694Crossref PubMed Scopus (55) Google Scholar, 17Bujalowski W. Klonowska M.M. J. Biol. Chem. 1994; 269: 31359-31371Abstract Full Text PDF PubMed Google Scholar). The concentration of the protein was spectrophotometrically determined, using extinction coefficient ε280 = 1.85 × 105cm−1m−1 (hexamer) (7Bujalowski W. Klonowska M.M. Jezewska M.J. J. Biol. Chem. 1994; 269: 31350-31358Abstract Full Text PDF PubMed 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, generously provided by Dr. R. McMacken. The site-directed mutagenesis was accomplished in the NIEHS Center facility (National Institutes of Health) directed by Dr. T. Wood. Labeling of the 6 cysteine residues of the DnaB variant, R14C hexamer, with CPM was performed in H buffer (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 CPM/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 by applying the sample on a DEAE-cellulose column and eluting with buffer T2 containing 500 mm NaCl. The degree of labeling was determined by absorbance of the marker at 394 nm using the extinction coefficient of CPM, ε394 = 27 × 103 cm−1m−1, providing the value of 5.8 ± 0.1 of CPM per DnaB hexamer. 2S. Rajendran, M. J. Jezewska, and W. Bujalowski, manuscript in preparation. All nucleic acids were purchased from Midland Certified Reagents (Midland, TX). The etheno-derivatives of nucleic acids were obtained by modification with chloroacetaldehyde (12Bujalowski W. Jezewska M.J. Biochemistry. 1995; 34: 8513-8519Crossref PubMed Scopus (120) Google Scholar, 18Secrist J.A. Bario J.R. Leonard N.J. Weber G. Biochemistry. 1972; 11: 3499-3506Crossref PubMed Scopus (496) Google Scholar). Oligomer dT(pT)19, labeled at the 5′-end with fluorescein, 5′-Fl-dT(pT)19, was synthesized using fluorescein phosphoramidate (Glen Research). Labeling of the 3′-end was performed by synthesizing dT(pT)19 with the last residue at the 3′-end of the oligomer having the amino group on a six-carbon linker. The amino group was subsequently modified with fluorescein 5′-isothiocyanate to obtain dT(pT)19-Fl-3′. The degree of labeling was determined by absorbance at 494 nm (pH 9), using the extinction coefficient, 7.6 × 104m−1 cm−1 (13Jezewska M.J. Kim U-S. Bujalowski W. Biochemistry. 1996; 35: 2129-2145Crossref PubMed Scopus (82) Google Scholar). The same procedures were used for labeling the 5′- and 3′-ends of the dA(pA)9. The concentrations of labeled oligomers were spectrophotometrically determined at 260 nm (pH 8.1), using extinction coefficients, 1.76 × 105m−1cm−1 and 11.4 × 105m−1 cm−1, respectively (13Jezewska M.J. Kim U-S. Bujalowski W. Biochemistry. 1996; 35: 2129-2145Crossref PubMed Scopus (82) Google Scholar). The concentrations of dεA(pεA)9, dεA(pεA)8, dεA(pεA)7, dεA(pεA)6, dεA(pεA)5, dεA(pεA)4, and dεA(pεA)3 were determined using extinction coefficients 37 × 103, 33.3 × 103, 29.6 × 103, 25.9 × 103, 22.2 × 103, 18.5 × 103, and 14.8 × 103m−1 cm−1 at 257 nm, respectively (12Bujalowski W. Jezewska M.J. Biochemistry. 1995; 34: 8513-8519Crossref PubMed Scopus (120) Google Scholar, 13Jezewska M.J. Kim U-S. Bujalowski W. Biochemistry. 1996; 35: 2129-2145Crossref PubMed Scopus (82) Google Scholar, 19Ledneva R.K. Razjivin A.P. Kost A.A. Bogdanov A.A. Nucleic Acid Res. 1977; 5: 4226-4243Google Scholar). Labeling the 5′-ends of ssDNA oligomers with 32P was performed using the standard procedure (12Bujalowski W. Jezewska M.J. Biochemistry. 1995; 34: 8513-8519Crossref PubMed Scopus (120) Google Scholar). Analytical sedimentation experiments were performed using an Optima XL-A analytical ultracentrifuge. Analyses of the sedimentation runs were performed as we previously described (8Jezewska M.J. Bujalowski W. J. Biol. Chem. 1996; 271: 4261-4265Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 9Reha-Krantz L.J. Hurwitz J. J. Biol. Chem. 1978; 253: 4051-4057Abstract Full Text PDF PubMed Google Scholar, 13Jezewska M.J. Kim U-S. Bujalowski W. Biochemistry. 1996; 35: 2129-2145Crossref PubMed Scopus (82) Google Scholar). The reported values of sedimentation coefficients were corrected to standard conditions,s20,w, for solvent density and viscosity (7Bujalowski W. Klonowska M.M. Jezewska M.J. J. Biol. Chem. 1994; 269: 31350-31358Abstract Full Text PDF PubMed Google Scholar). All steady-state fluorescence measurements were performed using the SLM-Aminco 48000S and 8100 spectrofluorometers (20Jezewska M.J. Bujalowski W. Biophys. Chem. 1997; 64: 253-269Crossref PubMed Scopus (37) Google Scholar). The emission spectra were corrected for a wavelength dependence of the instrument response using a software provided by the manufacturer. The binding of the DnaB protein was followed by monitoring the fluorescence of the etheno-derivatives of ssDNA oligomers (λex = 325 nm, λem = 410 nm). All titration points were corrected for dilution and, if necessary, for inner filter effect using the formula (15Bujalowski W. Klonowska M.M. Biochemistry. 1993; 32: 5888-5900Crossref PubMed Scopus (104) Google Scholar), Ficor=(Fi−Bi)ViVo100.5b(Aiλex)Equation 1 where Ficor is the corrected value of the fluorescence intensity at a given point of titration i, Fi is the experimentally measured fluorescence intensity, Bi is the background, Vi is the volume of the sample at a given titration point, Vo is the initial volume of the sample, b is the total length of the optical path in the cuvette expressed in centimeters, and Aiλex is the absorbance of the sample at the excitation wavelength. Computer fits were performed using KaleidaGraph software (Synergy Software, PA) and Mathematica (Wolfram Research, IL). The relative fluorescence increase of the nucleic acid, ΔF, upon binding the DnaB protein is defined by the equation, ΔF=(Ficor−Fo)FoEquation 2 where Ficor is defined by Equation 1, and Fo is the initial value of the fluorescence of the same solution. All steady-state fluorescence anisotropy measurements were performed in the L format, using Glan-Thompson polarizers placed in the excitation and emission channels. The fluorescence anisotropy, r, of the sample was calculated by the equation, r=(IVV−GIVH)(IVV+2GIVH)Equation 3 where I is the fluorescence intensity, and the first and second subscripts refer to vertical (V) polarization of the excitation and vertical (V) or horizontal (H) polarization of the emitted light (16Bujalowski W. Klonowska M.M. Biochemistry. 1994; 33: 4682-4694Crossref PubMed Scopus (55) Google Scholar). The factor G = IHV/IHH corrects for the different sensitivity of the emission monochromator for vertically and horizontally polarized light (21Azumi T. McGlynn S.P. J. Chem. Phys. 1962; 37: 2413-2420Crossref Scopus (424) Google Scholar). The limiting fluorescence anisotropies of fluorophores,ro, were determined by measuring the anisotropy of a given sample at different solution viscosity, adjusted by sucrose or glycerol, and extrapolating to viscosity = ∞, using the Perrin equation (22Lakowicz J.R. Principle of Fluorescence Spectroscopy. Plenum Publishing Corp., New York1983: 111-339Google Scholar). The efficiency of the fluorescence radiationless energy transfer, E, from CPM (donor), located on the small 12-kDa domains of the DnaB protein variant R14C, to the fluorescein (acceptor), located at the 5′- or 3′-end of dT(pT)19, bound in the DNA binding site of the helicase, has been determined using two independent methods. The fluorescence of the donor in the presence of the acceptor, FDA, is related to the fluorescence of the same donor, FD, in the absence of the acceptor by the equation, FDA=(1−νD)FD+FDνD(1−ED)Equation 4 where νD is the fraction of donors in the complex with the acceptor, and ED is the average fluorescence energy transfer from donor to acceptor, determined from the quenching of the donor fluorescence. Thus, the average transfer efficiency,ED, obtained from the quenching of the CPM fluorescence upon binding of the labeled ssDNA oligomer, is obtained by rearranging Equation 4, ED=1νDFD−FDAFDEquation 5 where, in the considered case, FD and FDA are the values of the CPM fluorescence intensity in the absence and presence of bound 5′-Fl-dT(pT)19 or dT(pT)19-Fl-3′. The value of νD has been determined using the binding constants of the 20- and 10-mers for the DnaB helicase measured in the same solution conditions (13Jezewska M.J. Kim U-S. Bujalowski W. Biochemistry. 1996; 35: 2129-2145Crossref PubMed Scopus (82) Google Scholar). In the second independent method, the average fluorescence transfer efficiency, EA, has been determined, using a sensitized acceptor fluorescence by measuring the fluorescence intensity of the acceptor (fluorescein) excited at 435 nm, where the donor (CPM) predominantly absorbs, in the absence and presence of R14C-CPM. The fluorescence intensities of the acceptor in the absence,FA, and presence, FAD, of the donor are defined as follows, FA=IoεACATφFAEquation 6 and FAD=(1−νA)FA+IoεAνACATφBA+IoεDCDTνDφBAEAEquation 7 where Io is the intensity of incident light,CAT and CDT are the total concentrations of acceptor and donor, νA is the fraction of acceptors in the complex with donors, εA and εD are the molar absorption coefficients of acceptor and donor at the excitation wavelength (435 nm), respectively; φFA and φBA are the quantum yields of the free and bound acceptor; and EA is the average transfer efficiency determined by acceptor-sensitized emission. All quantities in Equations 6 and 7 can be experimentally determined. For the case considered in this work, the acceptor is practically completely saturated with the donor, i.e. νA = 1. Thus, for νA = 1, dividing Equation 7 by Equation 6 and rearranging provides the average transfer efficiency as described by the following. EA=1νDεACATεDCDTφFAφBAFADFA−1Equation 8 It should be pointed out that the energy transfer efficiencies,ED and EA, are apparent quantities. ED 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 EA is a fraction of all photons absorbed by the donor that were transferred to the acceptor. The true Förster energy transfer efficiency, E, is a fraction of 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 (22Lakowicz J.R. Principle of Fluorescence Spectroscopy. Plenum Publishing Corp., New York1983: 111-339Google Scholar). The value of E is related to the apparent quantities of ED and EA, by the following (23Berman H.A Yguerabide J. Taylor P. Biochemistry. 1980; 19: 2226-2235Crossref PubMed Scopus (62) Google Scholar). E=EA(1−ED+EA)Equation 9 Thus, measurements of the transfer efficiency, using both methods, are not alternatives but parts of the analysis used to obtain the true efficiency of the fluorescence energy transfer process,E. The fluorescence energy transfer efficiency between donor and acceptor dipoles is related to the distance, R, separating the dipoles by the equation, E=Ro6Ro6+R6Equation 10 where Ro = 9790 (κ2 n−4 φd J) 16 is the so called Förster critical distance (in angstroms), the distance at which the transfer efficiency is 50%; κ2 is the orientation factor; φd is the donor quantum yield in the absence of the acceptor; and n is the refractive index of the medium (n = 1.4) (22Lakowicz J.R. Principle of Fluorescence Spectroscopy. Plenum Publishing Corp., New York1983: 111-339Google Scholar). The overlap integral, J, characterizes the resonance between the donor and acceptor dipoles. The fluorescence transfer efficiency of chemically identical donor and acceptor pairs, characterized by the same quantum yields, depends on the distance between the donor and acceptor, R, and the factor, κ2, describing the mutual orientation of the donor and acceptor dipoles (22Lakowicz J.R. Principle of Fluorescence Spectroscopy. Plenum Publishing Corp., New York1983: 111-339Google Scholar). Although in the work presented in this paper we are interested in relative distances between donors and acceptors, evaluation of κ2 allowed us to estimate the effect of the orientation factor on the differences between the studied donor-acceptor distances. The factor κ2 cannot be experimentally determined; however, the upper (κ2max) and lower (κ2min) limits of κ2 can be obtained from the measured limiting anisotropies of the donor and acceptor and the calculated axial depolarization factors, using the procedure described by Dale et al. (24Dale R.E. Esinger J. Blumberg W.E. Biophys. J. 1979; 26: 161-194Abstract Full Text PDF PubMed Scopus (659) Google Scholar). When both axial depolarization factors are positive, κ2maxand κ2min can be calculated from κ2max = ( 23)(1 + <dXD> + <dXA> + 3<dXD><dXA>) and κ2min = ( 23)(1 − (½)(<dXD> + <dXA>), where <dXD> and <dXA> are the axial depolarization factors for the donor and acceptor, respectively (24Dale R.E. Esinger J. Blumberg W.E. Biophys. J. 1979; 26: 161-194Abstract Full Text PDF PubMed Scopus (659) Google Scholar). The axial depolarization factors have been calculated as square roots of the ratios of the limiting anisotropies of the donors (CPM on the DnaB helicase) and acceptors (fluorescein at the 5′- or 3′-end of the ssDNA oligomers) and their corresponding fundamental anisotropies (17Bujalowski W. Klonowska M.M. J. Biol. Chem. 1994; 269: 31359-31371Abstract Full Text PDF PubMed Google Scholar). For two chemically identical donor-acceptor pairs, characterized by the sameRo (the same κ2, φd, and J), the differences in the transfer efficiencies,E1 and E2, result exclusively from the different distances between the donor and acceptor, R1 and R2. The relative ratio of the two distances is then defined by using Equation10 as follows. R1R2=[(1−E1)E2][(1−E2)E1]1/6Equation 11 In this work, we followed the binding of the DnaB protein to the ssDNA oligomers by monitoring the fluorescence increase, ΔF, of ssDNA etheno-derivatives upon the complex formation. Proteins and nucleic acids may form complexes characterized by different spectroscopic properties, particularly when multiple ligand binding processes are studied. In applying spectroscopic methods to monitor the ligand macromolecule interactions, one should not assume strict proportionality between the observed signal change and the degree of binding unless the existence of such proportionality has been shown (15Bujalowski W. Klonowska M.M. Biochemistry. 1993; 32: 5888-5900Crossref PubMed Scopus (104) Google Scholar). The general method to obtain thermodynamically rigorous estimates of the average degree of binding of the protein per ssDNA oligomer, Σνi, and the free protein concentration,PF, has been previously described by us (8Jezewska M.J. Bujalowski W. J. Biol. Chem. 1996; 271: 4261-4265Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 15Bujalowski W. Klonowska M.M. Biochemistry. 1993; 32: 5888-5900Crossref PubMed Scopus (104) Google Scholar, 25Jezewska M.J. Bujalowski W. Biochemistry. 1996; 35: 2117-2128Crossref PubMed Scopus (84) Google Scholar). Briefly, the experimentally observed ΔF has a contribution from each of the different possible “i” complexes of the DnaB hexamer with a nucleic acid. Thus, the observed fluorescence increase is functionally related to Σνi by the equation, ΔF=∑νiΔFimaxEquation 12 where ΔFimax is the molecular parameter characterizing the maximum fluorescence increase of the nucleic acid with the DnaB protein bound in complexi. The same value of ΔF, obtained at two different total nucleic acid concentrations, NT1 and NT2, indicates the same physical state of the nucleic acid, i.e. the degree of binding, Σνi, and the free DnaB protein concentration,PF, must be the same. The value of Σνi and PF is then related to the total protein concentrations, PT1 and PT2, and the total nucleic acid concentrations, NT1 and NT2, at the same value of ΔF, by the following equations, ∑νi=(PT2−PT1)(NT2−NT1)Equation 13 PF=PTx−(∑νi)NTxEquation 14 where x = 1 or 2 (12Bujalowski W. Jezewska M.J. Biochemistry. 1995; 34: 8513-8519Crossref PubMed Scopus (120) Google Scholar, 20Jezewska M.J. Bujalowski W. Biophys. Chem. 1997; 64: 253-269Crossref PubMed Scopus (37) Google Scholar). Quantitative fluorescence titrations and photo-cross-linking experiments, using ssDNA oligomers, showed that the DnaB hexamer has a single ssDNA binding site encompassing 20 ± 3 nucleotide residues and located predominantly on a single subunit (12Bujalowski W. Jezewska M.J. Biochemistry. 1995; 34: 8513-8519Crossref PubMed Scopus (120) Google Scholar, 13Jezewska M.J. Kim U-S. Bujalowski W. Biochemistry. 1996; 35: 2129-2145Crossref PubMed Scopus (82) Google Scholar, 14Jezewska M.J. Kim U-S. Bujalowski W. Biophys. J. 1996; 71: 2075-2086Abstract Full Text PDF PubMed Scopus (41) Google Scholar). The first evidence of the structural heterogeneity within the DNA binding site came from nuclease digestion-protection studies of the DNA in the complex with the helicase. In the first set of experiments, the complex of the DnaB hexamer with the 20-mer dT(pT)19labeled at its 5′-end with 32P in the presence of 1 mm AMP-PNP was subjected to micrococcal nuclease digestion as a function of time. The protein was in molar excess over the 20-mer to ensure complete saturation of the nucleic acid. Fig. 1 a shows the polyacrylamide sequencing gel of dT(pT)19 after digestion with the nuclease, at different time intervals, in the absence and presence of the helicase. In the absence of the helicase, in our solution conditions, the 20-mer was digested within 20 min. A dramatically different behavior was observed in the presence of the enzyme. The digestion process was less efficient, indicating significant protection of the nucleic acid against the nuclease by the enzyme. Moreover, at prolonged digestion times, a nucleic acid fragment of 10 or 11 nucleotide residues was strongly protected by the helicase. At the longest times, this was the major nucleic acid fragment on the gel, resistant to further nuclease action (Fig. 1 a). The size of the protected fragment was not dependent upon the length or type of base of the oligomer bound to the DnaB protein, indicating that protection against the nuclease digestion is limited to the nucleic acid bound within the single DNA binding site of the helicase. Fig. 1 b shows polyacrylamide sequencing gels of dA(pA)69 after digestion with the nuclease, at different time intervals, and in the absence and presence of the helicase. As in the case of dT(pT)19, the only predominant oligomer protected by the helicase in the complex with dA(pA)69, after prolonged digestion, is a ssDNA fragment, 10 or 11 nucleotide residues long. These data indicate that, within the total DNA binding site of the DnaB helicase, approximately half of the ∼20 nucleotide residues occluded by the helicase are bound differently than the remaining half, resulting in the observed nuclease digestion pattern. Thus, these results indicate that the total DNA binding site of the DnaB helicase is built of two structurally and possibly functionally different binding subsites (see below). To determine whether or not there is a difference in affinities between t" @default.
• W2003660437 created "2016-06-24" @default.
• W2003660437 creator A5025276977 @default.
• W2003660437 creator A5032509528 @default.
• W2003660437 creator A5073159818 @default.
• W2003660437 date "1998-04-01" @default.
• W2003660437 modified "2023-09-30" @default.
• W2003660437 title "Functional and Structural Heterogeneity of the DNA Binding Site of the Escherichia coli Primary Replicative Helicase DnaB Protein" @default.
• W2003660437 cites W1512516275 @default.
• W2003660437 cites W1515990643 @default.
• W2003660437 cites W1531702456 @default.
• W2003660437 cites W1537586850 @default.
• W2003660437 cites W1574146385 @default.
• W2003660437 cites W1605117055 @default.
• W2003660437 cites W1725095175 @default.
• W2003660437 cites W1983281161 @default.
• W2003660437 cites W1991190990 @default.
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