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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol . 261,No. Issue of July 5, pp. 8899-8907 1986 0 1986 by The American Swiety of Biological Chemists, Inc Printed in L~.s.A. Allosteric Conversion of Z DNA to an Intercalated Right-handed Conformation by Daunomycin* (Received for publication, December 9, 1985) Jonathan B. Chaires From the Department of Biochemistry, The University of Mississippi Medical Center, Jackson, Mississippi 39216-4505 Absorbance and fluorescence methods were used to minichromosome (5). Nuclear proteins that interact specifi- measure the binding the anticancer drug of daunomy- cally with Z DNA havebeen isolated from a variety of sources cin to poly(dGdC) under ionic conditions that initially (6-8). The precise role of Z DNA in gene expression, however, favor the left-handed Z conformation of the polymer. remains undefined. Recent reviews summarize the current Drug binding was cooperative under these conditions understanding of Z DNA structure, dynamics, and function and may be fully accounted for by an allosteric model (9-11). not in which the drugbinds preferentially (but exclu- A number of small molecules, among them the intercalators, sively) to the right-handed B conformation and shifts may have profound effects on the B to Z transition (12-17). the polymer from theZ to an intercalated right-handed Intercalators, for example, inhibit the rateof B to Z transition conformation. Quantitative analysis of binding iso- (13). In addition, intercalators may act as allosteric effectors therms in terms of the allosteric model allowed for and convert Z DNA to an intercalated right-handed form the estimation of the equilibrium constants for conver- sion of a base pair at a B-Z interface from the to the 2 under solution conditions that would otherwise favor the Z B conformation and for the formationof a base pair in conformation (12, 14, 16,17). Theexact mechanism by which the B conformation within a stretch of helix in the Z intercalators act as allosteric effectors on the B to Z equilib- conformation. The free energy the Z to B conversion of rium is poorly defined and is of interest not only in terms of of a base pair was calculated from thisdata and ranges the molecular mechanism of intercalator action, but in addi- from +0.03 to +0.3 kcal/mol over the NaCl range of tion in terms of the structure, stability, and dynamics of Z 2.4-3.5 M. The free energy for the formation a B-Z of DNA. Pohl and co-workers (12) first demonstrated striking junction was nearly constant +4.0 kcal/mol over the at allosteric effects of ethidium on Z form poly(dGdC) and used same range of NaCl concentrations. The salt depend- the allosteric model of Monod et al. (18) to analyze their ence of the free energy of the Z to B transition indicates cooperative binding data. While this model is certainly a preferential Na+ binding to the Z form and that there conceptually correct way to account for the observed cooper- is a net release of Na+ upon conversion of a base pair ative interaction of an intercalator with Z DNA, strict appli- from the Z to the B conformation. The energetically cation of this model neglects important aspects of the inter- unfavorable Z to B transition was found by this anal- calator-DNA interaction and of the B to Z equilibrium. In ysis to be driven by coupling to the energetically fa- particular, the phenomenon of neighbor exclusion (19, 20) is vorable interactionof daunomycin with B form DNA. not included in the model used by Pohl et al. (12) to analyze In 3.5 M NaCl, for example, the free energy changefor their data, nor is the B to Z equilibrium treated in a manner (Z + the overall reaction DNA base pairs) (daunomycin) appropriate for the structural transition of a DNA lattice. a (right-handed complex) is -7.0 kcal/mol, nearly all The allosteric model of Dattagupta et al. (21) is an extension of which is contributed by the binding of drug to B DNA. Analysis using the allosteric model also shows of the basic model of Monod et al. (18) that appropriately that the number of base pairs converted from theZ to treatsthe DNA structuraltransitionand which includes the B conformation per bound drug molecule is salt neighbor exclusion effects. The model of Dattagupta et al. dependent and provides evidence that drug molecules (21) is applied here to explain and analyze the cooperative partition into regions of the polymer in the right- interaction of the potent anticancer drug daunomycin with Z handed conformation. DNA. Previous results from this laboratory showed that dauno- mycin would effectively inhibit the rate of the B to Z transi- tion and would shift the midpoint of the NaC1-induced tran- The chemistryand biology of Z DNA, the left-handed sition of poly(dGdC) from the B to the Z form to higher salt helical form of DNA first observed in solution in 1972 byPohl concentrations (14). Further, under some conditions, the drug and Jovin (l), of intense current interest, in part because is would actively convert Z DNA back to an intercalated right- of the possible role of Z DNA in gene expression. Z DNA handed form (14, 17). The allosteric conversion of Z DNA to exists, or is inducible, in chromosomes from Drosophila (2,3) the intercalated right-handedform wasdemonstrated directly and Chironomus (4), and sequences that may adopt the Z over a range of ionic conditions by circular dichroism, sedi- conformation are found in the enhancer regions of the SV40 mentation velocity, and susceptibility to nuclease attack for both poly(dGdC) and poly(dGm'dC) (17). The results de- * This work wassupported by United States Public Health Service scribed here extend these previous findings. The allosteric Grant CA35635 awarded by the National Cancer Institute, Depart- model of Dattagupta et al. (21) is applied here to account for ment of Health and Human Services and by National Science Foun- dation Grant DMB-8421185. The costs of publication of this article the cooperative binding of daunomycin to DNA initially in were defrayed in part by the payment of page charges. This article the Z conformation and provides considerable insight into the must therefore be hereby marked "advertisement" in accordance with mechanism bywhich daunomycin converts Z DNA to an 18 U.S.C. Section 1734 solely to indicate this fact. intercalated right-handed form. Further, application of the 8899 8900 Allosteric Conversion of Z DNA model provides a description of the equilibriumof the Z t o B nz). Six parameters thus define the model. No attempt was made to transition over a range of NaCl concentrations, permitting use nonlinear regression to estimate these parameters since the six the estimation of the free energies of the formation of a €3-Z independent parameters preclude the usefulness of such an effort (21). The following algorithm was used to estimate values for the junction and for the conversion of a base pair at an interface parameters used in the allosteric model. For r values greater than the from the Z to the B conformation. The salt dependence of r value corresponding to themaximum in r/C, the polymer is entirely these transitions is also estimated. The model thus provides fit in the right-handed form, and data may be to the standard neighbor fundamental infor.mation o n the B t o Z transition in addition exclusion model as described above to estimate KB and ne. Kz may to providing a useful qualitative and quantitative theoretical be estimated from the intercept in the r/C axis, obtained by linear basis for understandinghow intercalators allosterically affect regression of low r data extrapolated to theintercept. KB, nB,and Kz may thus be estimated and constrained in the subsequent analysis. DNA conformation. From the statistics of the fits by this procedure KB, nB,and Kz are estimated to be accurate to 10-15%. The remaining parameters, nZ, MATERIALS ANDMETHODS s, and u were estimated by iterative adjustment.The value chosen for Poly(dGdC)-Poly(dGdC) (lot 317910)was purchased from P-L n~ had little effect on calculated isotherms, arising from the fact that Biochemicals. The sample was dissolved in a buffer containing 6m M there is little binding of the drug to the Z form. A value of nz = 2.0 Na2HP04,2 m NaH2P04, 1 m disodium EDTA, 0.185 M NaCl, was arbitrarily chosen, but increasing nz to 3.0 had no discernible M M pH 7.0. The dissolved sample was fractionated over Sepharose 4B, effect on the subsequent estimation of s and U . The parameters s and and thecentral fractionswere pooledand characterized as previously u are crucial in describing the low r region of the binding isotherm described (22). The sample was further characterized by sedimenta- with positive slope. The parameters s and KB/KZ determine the r tion velocity at 36,000 rpm in a Spinco model E analytical ultracen- value at the midpoint of this rising portion of the isotherm, and u trifuge equipped wi a photoelectric scanner system and electronic determines the steepness of the intraconversion of Z form to B form % speed control. The G ) method developed by van Holde was used to DNA. Values for s were found to be constrained to a rather narrow characterize the polydispersity of the sample (23). The sedimentation range in order to describe the experimental data. The nucleation rate at the midpoint of the boundary was 9.64 f 0.2 S; 90% of the parameter, u, was found to have a larger latitude. For example, data boundary sedimented within their range 9-10.4 S. These sedimenta- obtained a t 3.0 M NaCl could be described equally well by (a = 0.01, tion rates correspond to an average size of 1300 f 300 bp for the s = 0.75) or (u = 0.001, s = 0.73) with the remaining values fixed. sample (24). An extinction coefficient of 16,800 M” (bp’) cm” at 255 Our estimates for u thus are probably good to anorder of magnitude, nm was used for concentration determination (25). but values for s are defined to a much narrower range, probably within Measurement of the B to 2 Transition-The B to Z transition was 10%. For physical reasons, to be discussed later, u = 0.001 seems to measured using an individual sample for each NaCl concentration. A be the most reasonable choice. 1-ml sample (35.7 PM bp) was brought to the desired NaCl concen- Care was taken to test the validity of estimated values for the tration by the addition of solid NaCl and then dialyzed against a parameters for the allosteric model by additional experiments in lieu buffered NaCl solution of the desired concentration for a t least 24 h of rigorous statistical analysis. Thus, independent experiments were at room temperature. Followingdialysis, the complete UV absorbance conducted that yielded an estimate for KB/Kz,a prediction for the spectrum and the UV derivative absorbance spectrum were recorded salt concentration when s = 1.0, and the binding ratio required for for each sample a t ambient temperature using a Cary 219 spectro- the complete conversion of the polymer from the Z to theB form. All photometer. The ratio of absorbance at 260 nm to that at 295 nm of these were in excellent quantitative agreement with calculations was used as a measure of the B to Z transition (1). using the allosteric model with the parameters derived by the methods Binding Studies-Daunomycin (Sigma) binding to poly(dGdC) was described above. Without question, the parameters settled upon rep- measured using visible absorbance and fluorescence methods previ- resent one physically meaningful set. ously described in detail (14, 17, 22, 26). Poly(dGdC) samples for CompetitionDialysis-Competition dialysis measurements used binding studies were brought to the desired NaCl concentrations by the procedure of Mueller and Crothers (27). Samples of poly(dGdC) the addition of solid NaCl, followed by dialysis for 24-48 h against and calf thymus DNA were equilibrated to 3.5 M NaCl by dialysis buffered NaCl solutions of the desired concentrations. Titration and brought to an identical concentration of 80 pM (bp) by dilution experiments required 45-60-min equilibration times following the with dialysate. Under these conditions, the poly(dGdC) adopts the Z addition of drug because of the slow equilibration of the Z to B conformation, while the calf thymus DNA remains in the right- equilibrium. Following the addition of drug to poly(dGdC) in the Z handed conformation. Samples of poly(dGdC) and of calf thymus form, the absorbance or fluorescence was continuously monitored at DNA of identical volume and concentration were then dialyzed an appropriate fixed wavelength to ensure complete equilibration of against a common solution containing daunomycin and 3.5 M NaCl the binding reaction. until dialysis equilibrium was reached. The amount of free dauno- Data Analysis-Binding data were cast into the form of a Scatchard mycin in the dialysate and the amount of total daunomycin in the plot of r versus r/C, where r is the ratio of bound drug to the total respective polymer samples were then determined by fluorescence base pair concentration and C is the concentration of free drug. For intensity measurements (Aex = 480 nm; Lm= 555 nm), with reference concave binding isotherms, data beyond the r value corresponding to to a standard curve of daunomycin alone, following the addition of the maximum in r/C were fit to theneighbor exclusion model dimethyl sulfoxide to a concentration of 50% (v/v) to dissociate the drug-DNA complex (28). r/C = K(l - nr)((l - nr)/(l - (n - l)r))(”-l) RESULTS AND ANALYSIS~ where K is the binding constant to an isolated site and n is the neighbor exclusion parameter (19, 20). Anonlinearleast squares Cooperative Binding of Daunomycin to Z Form fitting routinebased on the Marquart-Levenberg algorithm available poly(dGdC)-Binding isotherms forthe interaction of dauno- through the National Institutes of Health PROPHET Computer mycin with poly(dGdC) under ionic conditions that initially Resource was used to fit the data. favortheleft-handed Z conformation of the polymer are Binding isotherms were “fit”to theallosteric model of Dattagupta et ~ l(21) by an iterative process using a computer program developed . shown in Fig. 1. In all cases shown, the plots are concave- by Prof. Donald Crothers and kindly made available by him. Briefly, down, indicative of a cooperative binding process. The allo- the model postulates that DNA is in equilibrium between two confor- steric binding model of Dattagupta et al. (21) may be used to mational states, which for the case a t hand may be unambiguously assigned to the B and Z conformations. An equilibrium constant, s, Portions of this paper (including part of ‘Results” and Figs. 1M- is assigned for the conversion of a base pair a t a B-Z interface from 4M) arepresented in miniprint a t the end of this paper. Miniprint is the Z to the conformation. An equilibrium constant, g s , is assigned B easily read with the aid of a standard magnifying glass. Full size for the formation of a base pair in the B conformation within a photocopies are available from the Journal of Biological Chemistry, stretch of helix in the Z conformation. Drug molecules may bind to 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. either the B or the Z form with characteristic binding constants (KB 85M-3992, cite the authors, and include a check or money order for and Kz, respectively) and characteristic neighbor exclusion (ng and $2.80 per set of photocopies. Full size photocopies are also included is in the microfilm edition of the Journal that available from Waverly The abbreviation used is: bp, base pair(s). Press. Allosteric Conversion of Z DNA 8901 analyze the binding data shown in Fig. 1. Table I shows the = -0.91, indicates that sodium ions are released as base pairs results of the analysis of the data in Fig. 1 in terms of the are converted from the Z to theB conformation. allosteric model. Calculated binding isotherms are indicated The data of Fig. 2 may be extrapolated to the point where in Fig. 1 by the solid lines. Excellent agreement between the s = l(ln s = 0) to obtain a corresponding sodium activity that experimental data and the allosteric model may be obtained is equivalent to [NaCl] = 2.3 M. This should correspond to with the parameters in Table I. the midpoint of the B to Z transition curve. Fig. 3 shows that Does Daunomycin Associate with Z DNA?-The methods this is in factthe case. The same poly(dGdC) sample used for used to obtain binding data assume that the observed changes the binding studies was used to obtain the dataof Fig. 3, and in the optical properties of daunomycin arise primarily from the resultant midpoint is in excellent agreement with that its intercalation into B form DNA. In light of recent results predicted by the extrapolation of the data of Fig. 2, as indi- that conclude that ethidium may intercalate intoZ form DNA cated by the solid symbol. This provides further independent with optical changes similar to those observed for its inter- verification of the reliability to theparameters estimated for calation into B form DNA (29), this assumption was critically the allosteric model. evaluated in a series of experiments that are described fully Sult Dependence of Ks and KZ-The salt dependence of the in the appended Miniprint. These experiments validate the intrinsic binding constants for the interaction of daunomycin assumption that theoptical changes observed in our binding with B and Z form DNA is shown in Fig. 4 The salt depend- . studies arise primarily from intercalation of the drug into B ence of daunomycin binding to Z form DNA is seen to be form DNA and indicate that theweak binding of the drug to greater than that seen for the interaction of the drug with B Z form DNA is accompanied by little change in the absorbance form DNA.For the interaction of the drug with B form DNA, and fluorescence properties of the drug. the slope of the line in Fig. 4 is -0.67. The slope of the line The relative affinity of daunomycin for B and Z form DNA for the interaction of the drug with 2 form DNA is nearly was tested directly by the method of competition dialysis (27). twice that value, -1.5. The differences in the slope must The results of the experiment are shown in Table 11. Dauno- reflect differences in the number of counterions associated mycin binds preferentially to the right-handed DNA, as is per phosphates between the B and Z conformations, as will evident from the higher total drug concentration that parti- be discussed in detail in a later section, suggesting greater ion tions into the calf thymus DNA samples. The ratio of bound binding to theZ conformation. daunomycin to the right- and left-handed DNA samples is The Number of Base Pairs Converted from the Z to the B calculated to be 40-50. In the limit as r approaches zero, that Conformation per Bound Drug Molecule-The allosteric ratio represents the ratio of the binding affinity of the drug model enables one to calculate the fraction of the polymer to right-and left-handed DNA (27). The value of 40-50 converted from the Z to the B conformation for each bound obtained in these experimentsis in excellent agreement with drug molecule. Fig. 5 shows the results of these calculations that calculated from the binding experiments in Fig. 1 (Table using the parameters shown in Table I. Circular dichroism I) and provides independent verification of the results ob- experiments (described in the Miniprint) at 2.4 and 3.5 M tained from the fits to the allosteric model. NaCl provided an independent approach to estimate the frac- Salt Dependence of the B to Z Transition-The data of tion of helix in theright-handed form as a function of bound Table I provides fundamental informationon the saltdepend- daunomycin. The agreement with the predictions from the ence of the Z to B transition. Fig. 2 shows the saltdependence allosteric model is excellent, lending confidence to the validity of the equilibrium constant for the Z to B transition as a in of those estimated parameters Table I. Total conversion to double logarithmic plot. The slope of the line, (d In s/d In a) the (intercalated) B form occurs at rcrit, which corresponds to 10.0 8.0 ; 6.0 I 4.0 FIG. 1. Interaction of daunomycin with poly(dGdC) underionic condi- 2.0 tions that initially favor the Z con- formation. Binding isotherms are shown for the binding of daunomycin to poly(dGdC) in solutions containing 3.5 M ( A ) ,3.0 M (B),2.8 M (c), 2.4 M (D) or NaCl. Binding data were obtained by absorbance and fluorescence methods. 10.0 The solid lines are calculated isotherms using the allosteric model described in 18.0 8.0 the text and the parameters listed in Table I. In panel D,the solid line is the 14.0 6.0 ? least-squares fit of the data beyond r = P la 0.05 to the neighbor exclusion model, la 10.0 while the dashed line is the isotherm 0 4.0 c calculated using the allosteric model. 8.0 2.0 2.0 0 0.1 0.2 0.3 0.4 r 8902 Allosteric Conversion of Z DNA TABLE I Summary of the parametersdescribing the allosteric binding of daunomycin to poly(dGdC) under ianie conditions favorable to the 2 conformation The parameters shown here were used to calculate the binding isotherms shown in Fig. 1. KB is the binding constant for the inter- action of a drugmolecule with an isolated site in the B conformation; n is the corresponding neighbor exclusion parameter. K z is the binding constant for the interaction of a drug molecule with an isolated site -0 1 0 2.0 3.0 4.0 . in the Z conformation. The corresponding neighbor exclusion param- eter, nZ, was set to 2.0 for all salt concentrations. The parameters NaC1, M describing the Z to B conformational transition are s, the equilibrium constant for the conversion of a base pair at a pre-existing B-Z FIG. 3. Salt-induced transition of poly(dGdC). The ratio of interface from the 2 to the B conformation, and u, the nucleation absorbance at 260 nm to that at 295 nm is shown as a function of parameter for the conversion of a base pair within a stretch of Z helix NaCl concentration. Poly(dGdC) at a concentration of 35.7 p~ (bp) to the B Conformation. was dialyzed at ambient temperature against a buffer containing 6 NaCl KB n Kz KdKz s 0 M M m Na2HP04,2 m NaH2P04,1 m Na2EDTA, pH 7.0, and NaCl M as indicated. Complete UV spectra and UV derivative spectra were M XIO-' M" bp XIO" M" recorded for each sample at 25 "C following dialysis. The symbols are Daunomycin the average of three determinations, and theerror bars represent the 2.4 3.2 2.8 1.8 17.5 0.95 0.001 range of the threereplicate determinations. 2.8 2.45 2.5 0.98 25.0 0.81 0.001 3.0 2.4 2.0 0.8 30.0 0.73 0.001 3.5 2.25 2.0 0.5 45.0 0.59 0.001 Adriamycin 2.8 8.8 2.0 1.06 88.0 0.85 0.001 TABLE I1 Summary of competition dialysis experiment M M Solution conditions were BP buffer (6 m Na2HP04,2 m H2P04, + pH 7.0) 3.5 M NaC1. Total polymer concentration was 80 FM (bp). Samples were dialyzed for 48 h. Dialysis equilibrium was complete as judged by control samples in which calf thymus DNA solutions were dialyzed against identicalcalf thymus DNA solutions. The totaldrug concentration within the dialysis sack was, in these cases, identical to within +5%. Bound daunomycin 0.4 0.6 0.8 1.0 I at n FIG. 4. Ionic strengthdependence of KBand Kz. the nat- A, ural logarithm of K g , the binding constant for the interaction of PM PM daunomycin with an isolated base pair in the B conformation, is 0.08 1.78 40.5 0.044 plotted as a function of the logarithm of the mean molal activity of 3.38 0.12 52.8 0.064 sodium, following Record et a . (36). The line is the linearleast l squares fit to the data, yielding a slope of -0.67 and a correlation coefficient of 0.903. B, the natural logarithm of Kz, the binding constant for the interaction of daunomycin with an isolated base pair in the Z conformation, as a function of the natural logarithm of the mean molal activity of sodium. The line is the linear least-squares fit 0 to the data, yielding a slope of -1.5 with a correlation coefficient of 0.955. - b C -0.2 the r value where the maximum in r/C occurs. The number of base pairs converted to theB conformation/drug molecule is 1/rC+ Table I11 summarizes the saltdependence of the rcrit, -0.4 and it is reciprocal. The data further emphasize the long range allosteric effects exerted by daunomycin on theDNA confor- mation. Nearly 19 bp are converted from the Z to the B conformation by the binding of a single drug molecule in 2.4 0.4 0.8 0.8 1.0 M NaC1, corresponding to nearly two turns of the helix. These In Q I effects are well beyond the immediate binding site of the drug, which spans 3-4 bp. FIG. 2. Ionic strengthdependence of the Z to B equilibrium Partitioning of Drug Molecules into Right-handed Regwm- constant (e). The natural logarithm of s, the equilibrium constant for the conversion of a base pair at a jundion from the 2 to the B The allosteric model may also be used to calculate the distri- conformation, is plottedas a function of the naturallogarithm of the bution of bound drug between the regions of the polymer in mean molal sodium activity. The open symbols are data taken from the B and Z conformations over the course of the titration. Table I. The solid line is the linear least-squares fit to the data, Fig. 6 shows the results of these calculations that provide yielding a slope of -0.91 (correlation coefficient = 0.9983). The solid evidence that drug molecules partition into the regions of the symbol is the experimentally determined midpoint of the B to Z polymer in the right-handed conformation. The plot shows transition (Fig. 3), at which point Ins = 0. The dashed line is the extrapolation of the fitted line and indicates that the midpoint is the binding ratio of drug within the regions of the polymer in correctly predicted by the fit to the data, independently verifying the the right-handed conformation as a function of the overall estimates for s i Table I. n binding ratio. The r value of drug in regions of the polymer Allosteric Conversion of Z DNA 8903 I I I in the right-handed conformation depends upon the total NaCl concentration andis nearly constant over a wide range of total bound drug. In 3.5 M NaC1, the binding ratio is near 0.3 mol of drug/molofbp,which correspondsto 1 drug molecule/3 bp in these regions, near the saturation level for of the drug. This indicates that most the drug is bound in the right-handed regions. A similar phenomenon occurs at the lower saltconcentrations,but witha lower bindingratio within the regions in the right-handed conformation, arising 0.1 0.2 0.3 from the fact that each bound drug molecule converts a longer stretch of helix to the right-handed conformation at these r bound ionic strengths. FIG. 5. Fraction of polymer in the €3 form as a function of Comparison with Adriamycin-The interaction of adria- bound daunomycin. The allosteric model was usedto calculate the mycin with poly(dGdC) in 2.8 M NaC1 was studied by the fraction of the polymer in the right-handed B form as a function of same methods as described above for daunomycin. Adriamy- the bindingratio r (= mol of daunomycin bound/molof bp) using the cin bindscooperatively to thepolymer under these conditions parameters in Table I. The lines refer, from left to right, to 2.4 M but converts poly(dGdC) to the right-handed form more effi- (-), 2.8 M (---), 3.0 M (-- - -), and 3.5 M (-----) NaC1. The ciently than daunomycin (data not shown). Adriamycin con- at symbols referto experimentally determined values 2.4 M NaCl (0) Z and 3.5 M NaCl (m) obtained by circular dichroism as described in verts 7.5 bp from the to theB conformation per bound drug the Miniprint. molecule, compared to 6.3 for daunomycin (Table 111). Anal- ysis of the adriamycin-poly(dGdC) interaction in terms of the allosteric model yields the parameters shown in the last line inTable I. Thesedataindicatethatadriamycin is more TABLE 111 efficient than daunomycin solely because of its higher affinity for B form DNA. Number of base pairs conuerted from the 2 to the B conformation/ bound daunomycin molecule DISCUSSION Values for rest, the binding ratio at which the polymer is converted entirely to the right-handed conformation, were calculated usingthe Daunomycin binds cooperatively to poly(dGdC) under ionic allosteric model as showninFig. 5. The reciprocal, l/rcfiL, the is conditions thatfavor the left-handed 2 form of the polymer. number of base pairs converted per bound drug molecule from the ZPrevious physical studies from this laboratory demonstrated to the B conformation. DM, daunomycin. - that daunomycin binding under these conditions converts the rhl vrctit polymer from the left-handed form to an intercalated right- Nacl (DM/bp) (bp/DM) handed form (17). Striking long-range allosteric effects are M apparent under certain conditions, with as little as 1 bound Daunomycin 18.5 2.4 0.054 drug molecule/20 bp driving the left to right conversion to 2.8 6.3 0.159 4.3 0.233 3.0 completion (17). The allosteric model of Dattagupta et al. (21) 3.5 3.3 0.303 to is used in the present report analyze the cooperative inter- action of daunomycin with Z DNA. The model may be used Adriamycin 2.8 0.133 7.5 to calculate binding isotherms that are in excellent agreement with the experimental results. The analysisprovides estimates for the equilibrium constants for the Z to B conversion over for a range of ionic strengths in addition to estimates binding constants and neighbor exclusion parameters describing the interaction of daunomycin with B and Z form DNA. This analysis provides fundamental information on the mechanism by which ligands affect the stabilityof Z DNA and the origins of the long-range allosteric effectspreviouslyreported. In addition, fundamental information on the Z to B transition is obtained by the analysis,over a range of Na' concentration where more direct physical measurements would be hampered by the fact that the B to Z equilibrium isshifted largely toward the Z conformation, and the average physical proper- ties would reflect mostly the Z conformation. FreeEnergy of the Z to B Transition-Analysis of the binding data shown in Fig. 1 using the allosteric model pro- 0.1 0.2 0.3 vides estimates for the equilibrium constant (s) for the con- r bound (TOW) version of a base pair at a pre-existing interface from the Z FIG. 6. Partitioning of bound daunomycin molecules. The to the B conformation and for the equilibrium constant for binding ratio of daunomycinwithinregionsin the right-handed the formation of a base pair in the B conformation with a conformation as a function of the overall binding ratio is shown. stretch of base pairs in the Z conformation ( 2 s ) . These values Curves were calculated usingthe allosteric model and the parameters are listed in Table I and may be used t o calculate the free in Table I for 3.5 M (-1, 3.0 M (- - -), and 2.8 M (- - - -) NaCI. energy for the transition of a base pair from the Z to the B Over a large range of total bound drug, the binding ratio within the conformation (AGO = RTln(s))and for the nucleationprocess right-handed regions is constant, indicating that the drug molecules are clustered. In all cases, binding ratio of drug within regions in (AGO = -RZ'ln(u2s)). T h e calculated values are listedin Table the the Z conformation is well over an order of magnitude lower than the IV. Over the NaCl concentrations studied, nucleation, with binding ratio of drug withinthe right-handed regions. the concomitant formationof two B-Z junctions, is energeti- 8904 Allosteric Conversion of Z DNA TABLE IV binding of the drug to B form. The difference between the Free energy of the Z to B transition free energy of drug binding to the Z and the B form is -2.3 The free energy for conversion of a basepair at aB-Z interface the kcal/mol and is the coupling free energy of the system. The from the 2 to the B conformationwas calculatedfrom the equilibrium negative sign indicates a favorable coupling free energy and constant s shown in Table I according to the equation A G h = indicates that the conversion of a base pair to the B confor- “‘Ins. The free energy for nucleation, the formation of a base pair in theB conformation within stretch of helix in the 2 conformation, a mation facilitates drug binding. is AGOnuelsstion -RTln$s. Two B-2 interfaces are created per nuclea- = Salt Dependence of the Z to B Tramition-The salt de- tion event. pendence of the equilibrium constant for the conversion of a base pair from the Z to the B conformation is shown in Fig. kcal/mol 2. The salt dependence arises from the linkage between so- M kcallmol 2.4 f0.03 +8.2 dium binding and the Z to B transition. The negative slope 2.8 +0.12 +8.3 seen in Fig. 2 indicates that the Z conformation is stabilized 3.0 +0.19 +8.4 is by increased sodium activity and that there a net release of 3.5 +0.31 +8.5 sodium ion upon conversion of the polymer to the B form. The saltdependence of the equilibrium constant s is governed by the equation P0B. POD (d In s/d In a,) = 2 A r Z where AI’& is the stoichiometrically weighted difference in preferential interaction parameters of the product and reac- tant macromolecular components (35). For the B to Z tran- sition, A G 2probably contains contributions from electrostatic = -7.3 and hydration effects, and itis, therefore, premature to assign detailed molecular meaning to the value of -0.91 found for the slope of the line in Fig. 2. The negative slope indicates unambiguously preferential sodium binding to Z form DNA. Further evidence for greater sodium ion binding to Z form DNA comes from the saltdependence of daunomycin binding FIG. 7. Free energy diagram for the interaction of dauno- to Z and B form DNA. Record and co-workers (36) have mycin with B and Z form DNA. The free energies for the inter- shown that for the interaction of a charged ligand with DNA, action of daunomycin with B and Z form DNA are shown schemati- cally following the suggestion Weber (34). The chemical potential of (dlnK/dln a*) = -23 +k of a base pair in the Z or B conformation is denoted as pz and p ~ , respectively. The chemical potential the freedrug is denoted as p ~ . of where Z is the charge on the ligand, \k is the fraction of The complexesofdaunomycinwith Z and B DNA basepairare counterions associated with each phosphate, and k is the net denoted p z and ~ B D .In this figure, the values for the equilibrium ~ amount of ion release from the ligand upon complex formation constants for the various steps were taken from the 3.5 M NaCl data in Table I and used to calculate the freeenergyaccording to the (36). In Fig. 3, the salt dependence of Kz is seen to be greater standard relationAG = -RTlnK. than the salt dependence of K g , with slopes of -1.5 and -0.67, respectively. Since the charge on daunomycin is expected to cally unfavorable, with a positive free energy of about 8 kcal/ be constant at +1 and there is no evidence for an appreciable mol. The free energy for the formation of a single B-Z junction contribution from k, this difference must arise from differ- is thus about +4 kcal/mol. The conversion of a base pair from ences in \k. Therefore, Z DNA must have a greater fraction the Z to the B conformation is characterized by a small of counterions associated per phosphate, or significant positive free energy. The signs and magnitudes of these free changes in the hydration of Z DNA occur upon ligand binding. energies are similar to those found for the B to Z transition Electrostatic calculations by Matthew and Richards (37) of poly(dGdC) inserts driven by supercoiling in low ionic show that Z DNA indeed binds more sodium ion and that Z strength (30-32). The dataof Table IV are ingood agreement DNA may contain specific sites for sodium binding in addition with the free energy estimates made by Pohl(33) for the B to to the more general electrostatic condensed ion-binding mode. Z transition using alternating GC oligonucleotides of defined A recent theoretical study by Soumpasis (38) predicts a salt- length. dependent free energy for the B to Z transition that is in good Energetics of the Allosteric Conversion of Z DNAto the agreement with that observed in Fig. 2. Intercalated Right-handed Form-Under the ionic conditions Origins of the Long-range Allosteric Effects-The extreme used in these studies, the conversion of poly(dGdC) from the cooperativity of the B to Z transition and the long-range Z to the B conformation is energetically unfavorable. The allosteric effects produced by daunomycin binding may be driving force for the conformational transition in the polymer understood as resulting from “incompatible” helix structures, i s provided by the coupling of the transition to theenergeti- as proposed as a general mechanism for the transmission of cally favorable binding of daunomycin to the B form of the conformational changes along the DNA helix by Crothers and polymer. This is shown schematically inthe free energy Fried (39). The free energy differences between the 2 and the diagram (34) in Fig. 7. The free energy for daunomycin B form of a base pair is slight (Table IV), but the formation binding to the B and Z forms is calculated from the binding of a boundary between the two forms is energetically unfa- constants in Table I using thestandard relation AG = vorable, with a free energy of over +4.0 kcal/mol. Because of -RTlnK. The free energy for the overall reaction the high free energy of the boundary, intermediates in which the two helical forms are intermixed will be avoided, and the (Z DNA base pair) + daunomycin e (daunomycin transition between the two helical conformations willbe sharp. Binding of a ligand to one of the helical forms, such as - B DNA base pair) the binding of daunomycin to the right-handed form, will lock is a favorable -7.0 kcal/mol, most of which comes from the that region of the helix into itsparticular conformation. This Allosteric Conversion of Z DNA 8905 altered structure will be propagated into adjacent regions of excess of Z DNA are nearly identical to those of the free drug. the helix in order to avoid the energetically unfavorable Since intercalationresultsina pronounced bathochromic boundary between the two helical forms. Ligands will tend to shift in the absorbance spectrum of the drug and in nearly partition into the regions containing itspreferred binding site, total quenching of the drug’s intrinsic fluorescence, the ab- a phenomenon indicated by Fig. 6. sence of pronounced optical changes suggest nonintercalative Comparisonwith Other Zntercalators-Walker et al. (16) binding under these conditions. Fluorescence quenching ex- have used the allosteric model to analyze binding data for the periments show that in the presence of excess Z form DNA, cooperative interaction of ethidium and actinomycin and ac- daunomycin is freely accessible to added quencher, again tinomone (16) with Z form poly(dGdC) in 4.4 M NaCl. Their suggestive of a nonintercalated complex. Second, the meas- results may be compared with those obtained in this study. ured binding constant for the interaction of daunomycin with The allosteric model provided an excellent fit to their exper- Z form DNA is lower in magnitude than what would be imental data. Ethidiumwas found to convert 3-4 bp from the expected for the formation of an intercalated complex. The Z to the B form per bound molecule, whereas actinomycin values for Kz in Table I are of comparable magnitude to the converted 5 bp. Under low salt conditions, in which 40 ~ L M equilibrium constant found for the first stepalong the reaction hexamine cobalt was used to convert the polymer to the Z pathway leading to the formation of a right-hand intercalation form, both ethidium and actinomycin converted 25 bp per complex (43). Since this step was interpreted asthe formation bound drug molecule to the right-handed form. These values of an “outside” bound form of the drug prior to the actual are of comparable magnitude to those reported here for dau- intercalation event, a similar type of complex is proposed here nomycin. The ratio of KB/Kz was found to be 300 for ethidium for the daunomycin-Z DNA interaction. The daunomycin-Z and 1000 for actinomycin. These appearlarger than the values DNA complex, then, is suggested to be a nonintercalated, found here for daunomycin and adriamycin but refer to a perhaps largely electrostatic, “outside” bound complex. Such higher sodium concentration. Extrapolation of the data of be a complex would freely accessible to added quencher, would Fig. 4 to 4.4 M NaCl predicts a KB/Kz ratio for daunomycin be mobile, and would be characterized by a low binding free of comparable magnitude tothat reported for ethidium. energy, all of which are in accord with the experimental Walker et al. (16) found that actinomone would not convert evidence reported here. A recent NMR study concludes that poly(dGdC) from the Z to theright-hand form in 4.4 M NaCl daunomycin binds exclusively to B form DNA (44). The data but would convert 11bp per bound drug molecule to the right- presented here show that daunomycin strongly prefers B form handed form under low salt conditions (16). This result is DNA but will form a weak complex with Z DNA. entirely consistent with studies on the B anomer of adria- Origin of Preferential Daunomycin Binding to B Form mycin (40), which show the same type of behavior. DNA-Daunomycin binds preferentially to right-handed Walker et al. (16) chose a value of a higher than used here DNA, as reflected by the KB/Kz ratios in Table I. The struc- to model their data. At 4.4 M NaC1, a = 0.013 and s = 0.56 turalbasis for this preference maybe inferred from the were used to generate an isotherm that matched their ethid- crystallographic data of Quigley et al. (45), in which the ium-binding data compared to a = 0.001, s = 0.59 used here strxture of a daunomycin-DNA complex was determined to to match data at 3.5 M NaCl. These two estimates for a are atomic resolution. Daunomycin was found to intercalate with probably within the tolerance of the iterative method used to the long axis of its anthraquinone ring system at right angles model the binding data, as discussed previously. There are, to the long axis of the DNA base pairs. The daunosamine however, significant physical reasons for believing that a is moiety and the acetyl group on the daunomycin A ring ex- of the order of First the free energy for the formation of tended in opposite directions to occupy the minor groove, two B-Z junctions is A O = -RTln($s). With a = 0.001, GJ providing anatural fit into the right-hand helix. Such a is 4.25 kcal mol”/junction at 3.5 M NaCl, a value that agrees favorable geometric fit would not bepossible in the left- with previous estimates of 3-5 kcal mol” (30, 31) and 5 kcal handed Z DNA. mol (32). The dataof Walker et al. (16) suggest a value of AGO Acknowledgment-I thank Prof. Donald Crothers for providing a = +2.7/junction, an estimate lower than those previously Fortran program for calculation of binding isotherms based on the reported. Second, a may be used to estimate thelength of the allosteric model and for his interest and insight into the problem. cooperative unit, No, at the midpoint of the B-Z transition according to the relation No = 1 + a+. For a = 0.001, No = REFERENCES 33 bp, while for a = 0.013, No= 10 bp. The cooperative length 1. Pohl, F. M., and Jovin, T . M. (1972) J. Mol. Bwl. 67,375-396 of the B-Z transition was experimentally estimated to be -25 2. Nordheim, A., Pardue, M. L., Lafer, E. M., Moller, A., Stollar, B. bp, corresponding to a = 0.0017 (41). Thus, a = certainly D., and Rich, A. (1981) Nature 294,417-422 represents a physically reasonable estimate consistent with 3. Lancillotti, F., Lopez, M. C., Alonso, C., and Stollar, D. (1985) J. several previous experimental observations. Cell Bwl. 100,1759-1766 4. Robert-Nicoud, M., Arndt-Jovin, D. J., Zarling, D. A., and Jovin, Nature of the Daunomycin-Z DNA Complex-The possibil- T. M. (1984) EMBO J. 3.721-731 ity of the intercalation of drugs into the Z DNA helix was 5. Nordheim, A., and Rich, A.-(1983)Proc. Natl. Acad. Sci. U. S. A . suggested on the basis of a stereochemical analysis by Gupta 80,1821-1825 et al. (42). Shaferand co-workers (29) subsequently proposed 6. Nordheim, A., Teaser, P., Azorin, F., Kwon, Y. H., Moller, A., that ethidium in fact intercalates inform poly(dGdC), based Z and Rich, A. (1982) Proc. Natl. Acad. Sci. U.S. A . 79, 7729- 7733 on experiments that indicated changes in the optical proper- 7. Azorin, F., and Rich, A. (1985) Cell 41,365-374 ties of ethidium in the presence of excess Z DNA that were 8. Lafer, E. M., Sousa, R., Rosen, B., Hsu, A., and Rich, A. (1985) similar to those observed upon its intercalation into right- Biochemistry 24,5070-5076 handed DNA. The proposal that ethidium intercalates intoZ 9. Rich, A., Nordheim, A., and Wang, A.H-J. (1984) Annu. Rev. DNA is contradicted by the early work of Pohl et al. (12) and Biochem. 53,791-846 the more recent work of Walker et al. (16). From the results 10. Leng, M. (1985) Biochirn. Biophys. Acta 826,339-344 11. Wells, R. D.(1985) in Progress in Clinical and Biological Research, of the studies described here, the formation of a daunomycin- Molecular Basis of Cancer (Rein, R., ed) Vol. 172A, pp. 47-53, Z DNA intercalation complex seems unlikely. First of all, the Alan R. Lias, Inc., New York optical properties of daunomycin in the presence of a large 12. Pohl, F.M., Jovin, T. M., Baehr, W., and Holbrook, J. J. (1972) 8906 Allosteric Conversion of 2 DNA Proc. Natl. Acad. Sci. U.S. A . 69, 3805-3809 Acad. Sci. U. s. A. 80,2447-2451 13. Mirau, P. A., and Kearns, D.R. (1983) Nucleic Acids Res. 11, 31. Klysik, J., Stirdivant, S. M., Singleton, C. K., Zacharias, W., and 1931-1991 Wells, R. D. (1983) J. Mol. Biol. 168, 51-71 14. Chaires, J. B. (1983) Nucleic Acids Res. 11,8485-8494 32. Peck, L. J., and Wang, J. C. (1983) Proc. Natl. Acad. Sci. U.S. 15. Chen, C., Knop, R. H., and Cohen, J. S. (1983) Biochemistry 22, A . 80,6206-6210 5468-5471 33. Pohl, F. M. (1983) Cold Spring Harbor Symp. Quunt. Biol. 47, 16. Walker, G. T., Stone, M. P., and Krugh, T. R.(1985) Biochemistry 113-117 24,7462-7479 34. Weber, G. (1975) Adu. Protein Chem.29, 1-83 17. Chaires, J. B. (1985) Biochemistry 24,7479-7486 35. Anderson, C. F., and Record, M. T. (1982) Annu. Rev. Phys. 18. Monod, J., Wyman, J., and Changeux, J-P. (1965) J. Mol. Biol. Chem. 33,191-222 12,88-118 36. Record, M. T., Jr., Anderson, C. F., and Lohman, T. M. (1978) 19. Crothers, D. M. (1968) Biopolymers 6, 575-584 Q. Reu. Biophys. 11,103-178 20. McGhee, J. D., and von Hippel, P. H. (1974) J. Mol. Biol. 86, 37. Matthew, J. B., and Richards, F. M. (1984) Biopolymers 23, 469-489 2743-2759 21. Dattagupta, N., Hogan, M., and Crothers, D. M. (1980) Bioehem- 38. Soumpasis, D-M. (1984) Proc. Natl. Acad. Sci.U. A. 81,5116- S. istry 19,5998-6005 5120 22. Chaires, J. B. (1983) Biochemistry 22, 4204-4211 39. Crothers, D. M., and Fried, M. (1983) Cold Spring Harbor Symp. 23. van Holde, K. E., and Weischet, W. 0.(1978) Biopolymers 17, Quunt. Biol. 47, 263-269 1387-1403 40. Britt, M., Zunino, F., and Chaires, J. B. (1986) Mol. Pharmacol. 24. Kovacic, R. T., and van Holde, K. E. (1977) Biochemistry 16, 29,74-80 1490-1498 41. Ivanov, V. I., and Minyat, E. E. (1981) Nucleic Acids Res. 9, 25. Wells, R. D., Larson, J. E., Grant, R.C., Shortle, B.E., and 4783-4798 Cantor, C. R. (1970) J. Mol. Biol. 54,465-486 42. Gupta, G., Dhingra, M. M., and Sarma, R. H. (1983) J. Biomol. 26, Chaires, J. B., Dattagupta, N., and Crothers, D. M. (1982) Bio- Struct. Dyn. 1,97-113 chemistry 2 1,3933-3940 43. Chaires, J. B., Dattagupta, N., and Crothers, D. M. (1985) Bio- 27. Muller, W., and Crothers, D.M. (1975) Eur. J. Biochem. 54, chemistry 24,260-267 267-277 44. Neumann, 3. M., Cavailles, J. A., Herve, M., Tran-Dinh, S., 28. Graves, D. E., and Krugh, T. R. (1983) Biochemistry 22, 3941- Langlois d'Estaintot, B., Huynh-Dinh, T., and Igolen, J. (1985) 3947 FEBS Lett. 182,360-364 29. Shafer, R. H., Brown, S. C., Delbarre, A., and Wade, D. (1984) 45. Quigley, G. J., Wang, A. H-J., Ughetto, G., van der Marel, G., Nucleic Acids Res. 12,4679-4690 van Boom, J. H., and Rich, A. (1980) Proc. Natl. Acad. Sei. U. 30. Singleton, C. K., Klysik, J., and Wells, R. D. (1983) Proc. Natl. S. A. 77, 7204-7208 Supplemental Material to The Allosteric Conversion of 2 DNA to an I n r e r e s l e t e d . Right-Handed Helix by Daunomycin Jonathan 8 . C h a i r e s ABSORBAXCE AN3 FLUORESCENCE PROPERTIES THE OF DAUNOIFICIN-Z DNA COWPLEX Yhile t h e r e is l i t t l e change in t h e relative f l u o r e s c e n c ei n t e n s i t y between daunomy- cin f r e e in Bolution and i n the presence of exeese 2 f o n DNA. t h e r e i8 11 measurable and t h a t the - A recent r e p o r t suggests t h a te t h i d i u m may intercalate i n t o L-form DNA. d i f f e r e n c e in t h ef l u o r t a e e n c ep o l a r i z a t i o n .I n 3.5 M NaC1, the f r e ed r u g shows B polari- int.irc(I1afianevent results i n o p t i c a l chengerr in ethidium similar t o rhoeoobservrdfor ration Of 0.133 + f - 0.004. while in t h e pre~ence of excess (DMfbp 0.012S) poly (dCdC). its b i n d i n gt o B-form DNA ( 2 9 ) . This o b s e m a t i o n 10 in c o n t r a d i c t i o n t o t h e earlier work t h e value increase* s l i g h t l y t o 0.160 +/- 0.003. Under t h e s e conditions. P value f o r t h e of P o h l e t d . (12). and the mare r e c e n t wcvk of Walker st. (16). Since theabeorbance d r u g i n t e r c a l a t e d into B f o m DNA cannot be d e t e m i n e d , aince t h e f l u o r e s c e n c e of t h e drug and f l u a r e e e e n c e methadauead in t h i a r e p o r t 10 a n a l y z e daunomycin binding t . I I B U ~ ~h a t The i. nearlycompletely quenched upon b i n d i n g to B fom DNA. The p o l a l i r l l t i o n of a dnunomy- i n Che drug arise from its interaction w i t h B form DNA, t h i s aSBumPtiOn o p t i c a lc h a n g e s d o - p o l y (dAdT) complex. which retains a p p r e c i a b l ef l u o r e a c e n c e ( 2 2 ) . was previaualyfound requires e x p e r i m e n t a l e r i f i c a t i o n . v Such data are preeenrsd here. The resulcs d e s c r i b e d t o be 0.6 for a polymer of approximatelythe 8816 size as rhepoly uaed (dMC) hare ( 2 2 ) . h e r e show that t h e n t e r a c r i o n i of daunomycin with 2 form DNA results i n little or no In presence the of exeella poly (dCdC). daunomycin t h u s rereins Lppreclsble rotational change in che abeorbance or f l u o r e s c e n c e o p e r t i e s pr of the Further. drug. iodide freedom. inconsistent w i t h what vauldheexpected for a drugintercslarad into DNA. quenchingexperiments show t h a t in the presence of a l a r g e molar excess of 2 DNA, daunomy- - cin 18 f r e e l y s c c e a r i b l e t o added queneher. end is t h e r e f o r e probablY nor i n t e r c a l a t e d . Figure M3 ahows a Stem-volmer p l o tf o tt h eq u e n c h i n g of daunomycin i n 3 . 5 M NaCl in Lhr prestlxe of e x ~ e s a (DM/bp 0 . 0 1 2 5 ) Z form poly (dCdC). The f r e ed r u g Y~II fovnd co - DNA. The extinction c o e f f i c i e n t of daunomycio is u n a l t e r e d i n the preaaoceof presence of The apparent extinetion c o e f f i c i e n t at 480 nm of daunomycin in 3 . 5 M NaCl in t h e exsees p o l y (dCdC) in t h e 2 farm or c a l f t h m s DNA in t h e B form i e *horn in excetls 2 have a Stern-Volmet quenching Constant. e e l l y are i n a e e o s s i b l s to added previously f a r daunomycin (22). and. quencher The s l o p e KBv, of Of l 13.4 U-' tho ine ( t h e r e f o r e .h a v e ). K BY in f i g u r e 13 p r o v i d e s K a" Incercelated - 12.5 0 ms indeed . typl- drug8 "(1. - found f i g u r e MI. Addition Qf low molar ratios of drug to B form DNA eeaulra i n am apparent +/- 0.3 M' -. daunomycin Thus. is f r e e l y acceaaible t o addedquencher in t h e presence Of e x t i n c t i o nc o e f f i c i e n t near 7000 M-lcm'l, B v a l u ei d e n t i c a l t o thatpreviouslydetermined e x c e s ~poly (dGdC). Contrary LO whatwould be expected i f ic vert i n t e r c a l s r e d . to b e h a r a c t e r i s t i c c the of t h e bound f o m of drug (26). ID Contrast, a d d i t i o n of IOU mler ratios of drug co Z form p e l y (dCdC) r e e u l ~ ein little L ~no change in thenppsrcnr I extinction c o e f f i c i e n t . A t a molar ratio of drug/bp of e t 0.01, t h a p p a r e ne x t i n c t i o n c o e f f i c i e n t is w i t h i n 5X of t h e value seen for t h e r u g d alone. These conditions ehould favor t h e fornation of even L weak complex, and t h e data suggest that t h e formation of B daunomycin 2 DNA c ~ m p l c xresulra in l i t t l e alteration of t h e absorbance of thedrug. This result contraat8 completely rho with reported result of Shsfer . 1 (29) f o r ethidium. and 1s consintent with the behaviot of ethidium reporred by Walker If.g. Pluoreseoce p r o p e r t i e s o f the daunomycin 2 DNA CODP~PX. F i g u r e M 2 ahows t h e r e l a t i v e f l u o r e s c e n c e intensity Of damomyein LIB a f u n c t i o n of total drug concentretion alone and i n the preaencc of exeees poly (dGdC) (L-form) and c a l f thymus DNA ( B form). In the of presence e x c e m B form DNA, daunomycin the intrineic f l u o r e s c e n c e of is nearly con- Allosteric Conversionof ZDNA CIRCULAR DICHROISM EXPERIHEWTS TO DETERMINE THE FRACTION OF RIGHT-HANDEU HELIX AS A fi P P A 11w. 1 N H C T I O N OF BO!lND DAIRIOHYCIN. Circular diehroiem (CO) was usad t o monitor the conformation of poly ( d M C ) 1111 a R function of bound daunomycinunder B formconditione(BPEbuffer) end LVO 2 form E w conditions (2.4 W NaCl and 3.5 I4 NaCl). CD titration experiments v c w perfomed T essentially (18 described in (16). CD spectra were recorded in B 1 cm p e t h l m g t h eel1 fhermoetsted et 2 . . 5C A Jasco 5-500 apectropolarimeter interfaced t o an 1BH PC computer E X wa8 uaed for all I L L ~ U ~ ~ ~ C ~ C Spectra B . yere recorded from 350 to 200 n at 0 2 nm m . T intervals, and four s p e c t r a were recorded and averaged for each sdditlonof drug. Wolar I N ellipticity "88 calculated, a f t e r baseline correction. according LO the equation, C T I [el - (LOO vhere C 18 the molar base p a i r concantrarion. 1 1 s the parhleogrh and 0 is the measured 0 N 6BBB. ellipticlLy. An Optical titration vas performed in parallel t o determine the binding r a t i o for each addition of daunomycin. 0. .I .2 . .4 .5 .6 .7 3 .8 0 I. <- O M H - 8 . P. W> A Figure H shows the results of the experiments. B form DNA ehovs an approximate inoellipric point at 295 nm (panel A ) . 2 f O r m DNA shows an inirially strang negative ellipticity ST this wavelength char increases with bovnd drug .e the polymer revert8 t o the right-handed conformation. At 250 nm (panel E) 2 DNA shows poairive ellipticity. while B form DNA shows II strong negative ellipticity. T e fraction of polymer (a) in the h Figure I44 from the equation. right-handed conformation nag be calculated from the data of a - 1 - ([e~,-rs~,)/(lel~-~e~~) vhere le], and [elg are the molar ellipticity of 2 and B form poly (dCdC) a t a given wavelength and le1 is the appnrtnr ellipticity at a particular binding ratio r . Figure 5 0 in che main t e x t shows the fraction o f right-handed hell,: as s function of t h e binding r a t i o r. 0 0 0 8 M 0 L A A A R E L L I T I C I T Y Figure W2. Fluorescence emission of daunomycin in the presence of B end 2 form DNA. The relative fluorescence intensity of dauamyycin in 3.5 II NaCl ia sham as P function of totel dlug eaneencration for the drug alone ( a ). in the presence of 80 YW (bp) poly (dcdc) ( 0 ) . and in the presence of 80 YW (bp) calf r h p u DNA (A). Tho fluorescence o f the drug in the preeenee of 2 form poly ( d M C ) is unchanged at low eoneentrationa of added M drug, but is nearly completely quenched the fluorescence measurements, l e x 2.5+ ... - nm. 480 in the presence of B form calf thymus DNA. hem I 555 n. m : . . . t . . . r . . . : . - . t lor 0 L A R 3. 2. k E L A / I A I P 0 T 0 I A A 0 I 9 T ~~ . . e. .1 .2 .3 .34 Figure 4 , W . I for the i n t e r a c t i o n of daunomycin with poly (dGdC). The symbole refer t o differcnt buffer eondiriana: (0) BPE (B form); ( 4 2.4 H NaCl ( 2 farm); ( b ) 3.5 n NaC1 ( 2 form). The data in panel A vas obtained at 295 W. while the datain panel B vas obtained LL 250 m n.
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