Thermodynamics and Kinetics of the Interaction of Copper (11) Ions With Native DNA

W. FORSTER, E. BAUER, H. SCHUTZ, and H. BERG, Ahademie der Wissenschaften der DDR, Forschungszentrum fur Molehularbiologie und Medizin, Zentralinstitut fur Mihrobiologie and experimentelle Therapie, Abteilung Biophysikochemie, DDR-69 Jena, GDR; and N. M. AKIMENKO, L. E. MINCHENKOVA, YU. M. EVDOKIMOV, and YA. M. VARSHAVSKY, Academy of Sciences of the U S S R , Institute of Molecular Biology, Moscow 11 7312, U S S R

Synopsis Based on equilibrium binding studies, as well as on kinetic investigations, two types of interactions of Cu2+ ions with native DNA a t low ionic strength could be characterized, namely, a nondenaturing and a denaturing complex formation. During a fast nondenaturing complex formation a t low relative ligand concentrations and a t low temperatures, different binding sites at the DNA bases become occupied by the metal ions. This type of interaction includes chelate formation of Cu2+ions with atoms N(7)of purine bases and the oxygens of the corresponding phosphate groups, chelation between atoms N(7)and 0 of C(6)of the guanine bases, as well as the formation of specific interstrand crosslink complexes a t adjacent G.C pairs of the sequence dGpC. CD spectra of the resulting nondenatured complex (DNA-CU~+),,~ may be interpreted in terms of a conformational change of DNA from the B-form to a C-like form on ligand binding. A slow cooperative denaturing complex formation occurs a t increased copper concentrations and/or a t increased temperatures. The uv absorption and CD spectra of the resulting complex, (DNA-Cu2+)denat,indicate DNA denaturation during this type of interaction. Such a conclusion is confirmed by microcalorimetric measurements, which show that the reaction consumes nearly the same amount of heat as acid denaturation of DNA. From these and the kinetic results, the following mechanism for the denaturing action of the ligands is suggested: binding of Cu2+ions to atoms N(3)of the cytosine bases takes place when the cytosines come to the outside of the double helix as a result of statistical fluctuations. After the completion of the binding process, the bases cannot return to their initial positions, and thus local denaturation at the G-C pairs is brought about. The probability of the necessary fluctuations occurring is increased by chelation of Cu2+ions between atoms N(7)and 0 of C(6) of the guanine bases during nondenaturing complex formation, which loosens one of the hydrogen bonds within the G-C pairs, as well as by raising the temperature. The implications of the new binding model, which comprises both the sequence-specific interstand crosslinks and the described mechanism of denaturing complex formation, are discussed and some predictions are made. The model is also used to explain the different renaturation properties of the denatured complexes of Cu2+,Cd2+, and Zn2+ ions with DNA. In temperature-jump experiments with the nondenatured complex (DNA-CU~+),,~,a specific kinetic effect is observed, namely, the appearance of a lag in the response to the perBiopolymers, Vol. 18,625-661 (1979) 01979 John Wiley & Sons, Inc.

0006-3525/79/00l8-0625$01.OO

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FORSTER ET AI,.

t u r h a t i o n . T h e resulting sigmoidal shape of t h e kinetic curves is considered t o he a comequence of'the necessity of disrupting a certain number of t h e crosslinks existing in t h e nondenatrirrd complex before t h e local unwinding of the hinding regions (a main s t e p o f denaiwin:: complex formation) may proceed.

INTKODUCTION T h e interaction of certain divalent transition metal ions such as Cu2++, %n2+,or Mn2+ with DNA and the possible role of these ions in biological processes form a field of permanent interest.' Some evidence has accumulated to suggest that specific nucleic acid-protein interactions may be mediated by such metal ions, which in some cases are found to be integral parts of important enzymes functioning with nucleic For this reason the formation of ternary complexes of Zn2+,Cu2+,and Mn2+ ions with both nucleic acid and protein residues has been studied by several authors.]:'-17 In this context a clear understanding of the mechanisms of interaction between these metal ions and the nucleic acid itself is highly desirable. Among the group of metal ions under consideration (Co2+,Ni", Mn", Zn", Cd", Cu"), the Cu2+ ions have the strongest abilit,y to bind with IINA:' and should be suited for model ions in investigations of binding mechanisms that could be of importance in other cases. A variety of methods has been applied in the past to elucidate the binding sites f'or Cu" ions in DNA.:'.18-2(j T h e d iculty, however, often arose of extrapolating the results obtained from studies with the nucleic acid components or with single-stranded polynucleotides to DNA.:3.27--:30 In order to explain certain important properties characteristic of the DNACu'+ system, several models have been suggested for the structure of DNA-Cu2+ c o m p l e x e ~ , 2(i' ~but ~ ~ none ~ has as yet been proven unambiguously. Further investigations are desirable to establish a more general binding model t h a t is capable of explaining all the known experimental facts. T h e conformational changes induced in native DNA by binding with Cu'+ ions were of special interest to us, because it has been suggested that copper ions might he able to regulate local DNA secondary s t r u c t u r e . : ' l ~ In a C1) study, the methodical aspect of which (application of matrix rank analysis) has been reported earlier,:j:jwe could discriminate between two distinct types of complexes formed on interaction of Cu2+ions with native

DNA. The aims of the present study were to characterize both types of complex formation in more detail by equilibrium methods, to investigate the kinetics of formation and dissociation of the complexes, as well as t o analyze the influence of the ligand binding on the kinetics of DNA denaturation. By this it was hoped to gain an insight into the mechanisms of interaction and to learn about the reaction steps and complex structures involved.

INTERACTION OF CU(I1) WITH NATIVE DNA

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EXPERIMENTAL Materials The DNA samples used were from Streptomyces chrysomallus (72 mol G C, molecular weight 14 X lo6, protein content 0.6%) and chicken erythrocytes (42 mol % G C, molecular weight 1.2 X 106, protein content 1in Fig. l ) , a slow reaction in the minute range is observed until CD spectra become constant. The result of a matrix rank analysis of 10 CD spectra of DNA-Cu2+ complexes with rt = 0-1, taken from the titration experiment with S. chrysomallus DNA, is presented in Fig. 2. Rank 4 was found for the spectra matrix.33 The fourth of these spectral components, located with its maximum a t 290 nm, was statistically less significant. Indeed, further analysis showed that two subsets of experimental spectra, each with rank

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& 2

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A Inml Fig. 1. CD spectra of DNA-Cu2+ complexes obtained on titration of native DNA from chicken erythrocytes (spectrum 0) with Cu2+ ions. Numbers on the spectra indicate the amount of Cu2+ions added, expressed by the ratio rt of total ligand to total DNA phosphate concentration. (a) Low rt range, (b) high rt range. Conditions: solvent 0.002M NaC1, t = 25"C, CDNA = 1.0 X 10-4M (P). Basic spectrum No. 2 (dots, see text) is included in (a).

INTERACTION OF CU(I1) W I T H NATIVE DNA

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Fig. 2. Experimental CD spectra and calculated CD basic spectra of DNA-Cu2+ complexes obtained from a titration series with S. chrysornallus DNA. Conditions: 0.002M KCI, t = 28OC, CDNA = 1.8 X 10-4M ( P ) . Spectrum 0: native DNA (basic spectrum No. 1);spectrum 0.3: DNA-Cu2+ complex with rt = 0.3 from the titration series (CD spectrum with minimal height of the positive band); spectrum 3: DNA-Cu2+ complex with rt = 3 from the titration series (CD spectrum a t saturation); spectrum (- -), DNA-Cu2+ complex with rt = 1.3, which has been heat denatured for 15 min at t = 60°C and then quickly cooled. 0 ,Basic spectrum No. 2; B, basic spectrum No. 3 (see text).

2, can be distinguished. Thus, besides the native DNA, mainly two complex structures are involved. The related complex formation processes occur without noticeable overlap, one a t low and the other a t high relative Cu2+concentrations. Because of the clear separation between these two processes, the forms of the initially unknown basic spectra Nos. 2 and 3, corresponding to the two complex structures, could be ~ a l c u l a t e d .In ~ ~the case of S. chrysomallus DNA, this was done assuming that at rt = 0.3, where the positive CD band reaches its minimal height, the first type of DNACu2+complex is formed completely. Thus the basic spectrum No. 2 was postulated to coincide with the experimental CD spectrum for rt = 0.3 a t one selected wavelength (270 nm). For the basic spectrum No. 3, on the other hand, zero amplitude a t 245 nm was postulated in order to compare it with the experimental CD spectrum of a heat-denatured DNA-Cu2+ complex. All three basic spectra (with the experimental CD spectrum of native DNA as basic spectrum No. 1)are given in Fig. 2, along with selected experimental CD spectra. Notice that the coincidence of basic spectrum No. 2 (dots) with the CD spectrum of the complex for rt = 0.3 is quite good, whereas marked devia-

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tions occur at longer wavelengths between basic spectrum No. 3 (squares) and the CD spectra of the DNA-Cu2+ complex at saturation (rt = 3) or of the heat-denatured DNA-Cu2+ complex included in Fig. 2. This is probably connected with the minor fourth spectral component mentioned, which, in our opinion, might be characteristic for the interaction with (G + C)-rich DNA. A t the moment, however, such a hypothesis cannot be proven unambiguously. In accordance with the above results, only one type of DNA-Cu2+ complex was found by matrix rank analysis of the low-range CD spectra [Fig. l(a),rt = 0-11 of the titration experiments with erythrocyte DNA. For calculating the corresponding basic spectrum, the CD amplitude at 290 nm was taken to be zero in order to test the similarity with the CD spectrum of DNA in the C-form (Li salt of calf thymus DNA in film at 75% rel. hum.44),which has crossing points at 290 and 275 nm. The resulting basic spectrum No. 2 [Fig. l(a), dotted line] also has a second crossing point at 275 nm and is very similar to the CD spectrum of DNA in the C-form. CD spectra resembling those of the DNA-Cu2+ complexes at low relative ligand concentrations have been observed for DNA in several other cases, e.g., in concentrated salt solutions, 4t-48 in ethylene glycol as ~ o l v e n t , 4 ~ , ~ ~ and in complexes with his tone^.^^.^^ The forms of these CD spectra are thought to be characteristic of a conformational transition from the B-form to a C-like conformation of DNA (transition within the family of B-conformations46). A more detailed comparison of some of the cases mentioned above is presented in Fig. 3, where several CD difference spectra are given (difference with respect to native DNA in the B-form). These spectra are normalized to the CD difference amplitude a t 270 nm, because only a comparison of their forms will be made. CD difference spectra of DNA-Cu2+ complexes, represented by basic spectra No. 2 for different DNAs, are shown together with CD difference spectra of DNA at different conditions: in high salt concentrations, in the C-form in film, and in nucleohistone. As can be seen, there arise deviations in the forms that are outside the experimental error. The basic spectra of the DNA-Cu2+ complexes, as well as of the C-form difference spectrum, have their maxima at 270 nm. [In the (G C)-rich DNA-Cu2+ complex, we observe shoulders at 260 and 285 nm, Fig. 3(b)]. The maxima of the CD difference spectra a t high salt concentrations, on the other hand, are located a t 275-280 nm. It is difficult to draw definite conclusions from the data of Fig. 3. Qualitatively, however, because of the difference mentioned in the position of the maxima, we are tempted to conclude a greater similarity of the C-form of DNA with the modified conformation in the DNA-Cu2+ complex than with the conformation at high salt concentration. From the CD results presented, it is not possible to establish a dependence of the ligand-induced conformational changes on the G C content of DNA. Because of the qualitative differences in the CD spectra of DNAs, an internal standard (namely,the CD spectrum of the C-form DNA in film)

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INTERACTION OF CU(I1) WITH NATIVE DNA

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Fig. 3. Comparison of the forms of selected CD difference spectra. (a) -0-,basic spectrum No. 2 of the DNA-Cu2+ complexes with erythrocyte DNA; -A-, calf thymus DNA in 8M LiCl (Ref. 46);. . .O.. ., calf thymus DNA in the C-form in film [Li salt a t 75% rel. hum. (Ref. 44)]; . . .A.. .,calf thymus nucleohistone in 2.5 X 10-3M Tris/HCl a t pH 7.5 (Ref. 52), isolated by the method of Maurer and Chalkley. (b) - 0 - ,basic spectrum No. 2 of the DNA-Cu*+ S. chrysornallus DNA in 8M LiCl (Ch. Zimmer, complexes with S. chr~sornallusDNA; -A-, personal communication). Ordinate: difference in CD amplitudes with respect to native DNA in the B-form, normalized to the value a t 270 nm.

+

would have to be used for an accurate estimation of such a G C dependence. Unfortunately, for (G C)-rich DNAs these C-form CD spectra have not yet been measured. The qualitative characteristics of the CD spectra indicate that within the first type of complex formation, corresponding to low relative Cu2+ concentrations (basic spectra No. 2), no DNA denaturation takes place. This conclusion is confirmed by uv absorption difference spectra measured under these conditions (Fig. 4): two isobestic points are observed around 238 and 260 nm a t small values of rt, along with a negative minimum a t 248 nm (hypochromic effect) and a broad positive band a t wavelengths greater than260nm. On the basis of the CD and absorption data presented so far, the complex formed between native DNA and Cu2+ ions a t low relative ligand concentrations will be called the nondenatured complex (DNA-CU~+),,~ in the following discussion. Microcalorimetric measurements showed that practically no thermal effect can be detected along with this type of complex formation (see below).

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A Cnml Fig. 4. The uv absorption difference spectra of DNA-Cu*+ complexes measured against native DNA. Titration of erythrocyte DNA with Cu2+ ions. Low rt range with values of rf indicated. Ordinate: difference in molar extinction coefficients A@). Conditions: 0.01M NaC1, t = 25"C, CDNA = 2.4 X 10-4M ( P ) .

A t increased ligand concentrations, on the other hand, a denatured complex (DNA-Cu2+)denatis formed. Indeed, a t higher values of rt [Figs. l ( b ) and 21, the CD spectra with a strongly decreased negative CD band are characteristic of DNA d e n a t ~ r a t i o n . The ~ ~ same is true for the absorption difference spectra (Fig. 5), which deviate from the isobestic points and become positive a t all wavelengths (hyperchromic effect). The position of the absorption difference maximum changes from about 290 to 270 nm, while the maximum of the DNA absorption band is shifted bathochromically from 259 to 261.5 nm.43 As mentioned earlier, this denaturing complex formation is a slow re-

INTERACTION OF CU(I1) WITH NATIVE DNA 3000

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Fig. 5. The uv absorption difference spectra of DNA-Cu2+ complexes in the high rt range. Spectra measured against native DNA after a reaction time of 2 hr at 30°C. Conditions: erythrocyte DNA, 0.01M NaCl, CDNA = 1.7 X 10-4M ( P ) . The difference spectrum for rt = 0.10 from Fig. 4 is included for comparison (dotted line).

action at low temperatures. For this reason the spectra of Figs. 5 and 6 have been measured 2 hr after addition of the ligand ions. Saturation of the spectral effects takes place in both absorption and CD spectra a t high values of rt (e.g., for rt > 5 in Figs. 5 and 6). The hyperchromism a t saturation of the complex (33% a t 260 nm in the experiment of Fig. 5) is comparable with that of heat-denatured DNA after cooling. In the experiment with S. chrysomallus DNA (Fig. a), where the denaturing complex formation took place between rt = 0.3 and 3, the hyperchromism (258 nm) a t these values of rt was 1and 37%, respectively. Qualitatively, as well as quantitatively, there is a close similarity of the absorption difference spectrum of the saturated complex (DNA-Cu2+)denatto that of the saturated complex DNAdenat-Cu2+(complex of heat-denatured and cooled DNA with Cu2+ ions; saturation of this complex was already reached a t rt 0.8 for erythrocyte DNA43). During the denaturing complex formation, a heat absorption could be measured by microcalorimetry. In the differential microcalorimeter used, the reaction between Cu2+ions and native DNA took place at conditions like those chosen in the experiments of Figs. 5 and 6. The results, namely, the reaction enthalpies of the endothermic complex formation a t 30°C, which depend on the relative ligand concentration, are given in Table I. At saturation a value of A H = (5.7 f 0.1) kcal/mol base pairs was found, which is near the value of AH = (5.9 f 0.1) kcal/mol base pairs measured for the same DNA a t acid denaturation (unpublished results).

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TABLE I Reaction Heat as a Function of Relative Ligand Concentration rt as Measured on Complex Formation of Native Erythrocyte DNA with Cu2+ Ionsa

Q

a

QIQsaturation

rt

(kcal/mol base pairs)

(%)

0.9 2.25 2.7 3.15 3.7 3.7 4.5 5.4 7.2

0.15 1.03 1.07 2.09 2.73 2.80 4.10 5.49 5.67

2.7 18.2 18.8 36.8 48.1 49.4 72.3 96.8 100

Conditions: 0.01M NaN03, t = 30°C, CDNA = 1.7 x 1 0 - 4 ~( P ) .

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A Inml Fig. 6. CD spectra of the DNA-Cu2+ complexes shown in Fig. 5.

Titration Curves and Binding Parameters To evaluate binding parameters (binding constant, size of binding sites, cooperativity), titration curves for the DNA-Cu2+ complexes were constructed from the spectral and microcalorimetric data. Figure 7 presents

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INTERACTION OF CU(I1) WITH NATIVE DNA I

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Fig. 7. Titration curves for complex formation of native erythrocyte DNA with Cu2+ions under various conditions. Curve 1: microcalorimetric data from Table I (0.01M NaN03, t = 30°C); curve 2: CD data a t 245 nm from Fig. 6 (0.01M NaC1, t = 30°C); curves 3a and 3b: CD data a t 280 and 245 nm, respectively, from Fig. 1 (0.002M NaCI, t = 25°C). Abscissa: negative logarithm of total Cu2+ion concentration. Ordinates: lower part, relative effects in percent, normalized to values a t saturation; upper part, CD amplitudes a t 280 nm.

such titration curves taken from the experiments with erythrocyte DNA. In curve 1the reaction heat, normalized to its value a t saturation, is given as a function of the total ligand concentration pCu:+ (on a logarithmic scale) at fixed DNA concentration. Using the same abscissa for the data of the titration experiments, the salt-dependent changes of the CD amplitude in the negative band a t 245 nm are shown by curves 2 and 3b (data from Figs. 6 and 1, respectively). Changes in the positive CD band at 280 nm on complex formation are given

FORSTER E T AL.

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by curve 3a (data from Fig. 1). Similarily, CD amplitudes a t two characteristic wavelengths (270 and 245 nm), taken from the titration experiment with S. chrysomallus DNA, are shown in Fig. 8. The fact that the two complexes (DNA-Cu2'),at and (DNA-Cu2+)denat are formed successively, established by matrix rank analysis, is quite clearly reflected in Figs. 7 and 8 by the curve corresponding to the positive CD band (280 and 270 nm, respectively). The changes in CD amplitudes have different directions in the low and the high rt ranges, and both these ranges are well separated. Spectrophotometric titration curves a t different temperatures derived from the kinetic experiments on complex formation with erythrocyte DNA (manual mixing) are shown in Fig. 9(a). The final difference absorption a t 275 nm, normalized to its maximal value a t saturation, is plotted as a function of pCut2+. The temperature dependence of the midpoints of these titration curves is indicated in Fig. 9(b). Note that a t a given value of rt, the extent of denaturing complex formation can be markedly increased by raising the temperature.

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INTERACTION OF CU(I1) WITH NATIVE DNA

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Fig. 9. Spectrophotometric titration curves for DNA-Cu2+ complex formation a t different temperatures. Conditions: erythrocyte DNA, 0.01M KCI, CDNA = 2.3 X 10-4M ( P ) . (a) Difference absorption vs native DNA a t 275 nm, normalized to values a t saturation. (b) Temperature dependence of half-transition points.

For the denaturing complex formation, which by the changes in the negative CD band is reflected almost exclusively, an evaluation of binding parameters was attempted. The suggestion to be tested was that there exists strong cooperativity, probably connected with some denaturation process occurring along with the ligand binding. From the steepness of binding curves like those in Figs. 7-9, where a quantity proportional to the degree of binding is given as a function of the logarithm of total ligand concentration, conclusions on cooperativity cannot be drawn directly. This would become possible with the free ligand concentration instead of the logarithm of total ligand concentration as abscissa, but the free copper concentration was not measured in our experiments. Another possible method of judging cooperativity, namely, from nonlinearities in Scatchard plots, is practicable only a t unity size of binding sites (the same holds for modified Scatchard plotss4). For these reasons, a nonlinear parameter fit of the experimental points was necessary. It was made for the CD data of Fig. 8(b),i.e., for the titration experiment with S. chrysomallus DNA, which was the subject of the matrix rank analysis presented earlier.

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To evaluate the binding parameters, we used the model of McGhee and von Hippel55 that describes the binding of ligands to a linear chain of monomer units, taking into account the nearest-neighbor interactions and the nonunity size of binding sites. The parameters of the model are the binding constant K for binding t o an isolated binding site (neighbor monomer units unoccupied), the size of binding sites a (in monomer units), and the cooperativity constant w (for binding to sites with one or both neighbor monomer units occupied the binding constants are K o or K o 2 , respectively). For the data-fitting procedure, the measured CD amplitudes A t = ( t ~ - t~ )245 nm were assumed to take the following form: At = A t p

+ ( A t p b - Acp)ar

Here A t p is the CD amplitude resulting from unoccupied nucleotides, Atpb is the CD amplitude resulting from nucleotides occupied by ligands that are bound in a denaturing manner, a is the number of nucleotides occupied by one Cu2+ion that is bound in a denaturing manner, and r is the ratio of the concentration CAb of bound ligands to the total concentration cpt of nucleotides. The connection between the quantities r, C A (concentration of free ligands), and the model parameters K , a , and o is given by the following formula (taken from Ref. 55 with an error in sign corrected):

r

-= K ( l

- ar)

CA

+r -R - 1)(1 - a r )

( 2 o - 1 ) ( 1- a r ) 2(w

with

R

= {[l- ( a

+ l)rI2+ 4wr(l - ar)j1/2

The relation between the free ( C A ) and total ( C A t ) ligand concentrations is given by the balance equation CAt = CA cptr. After programming the problem-specific subroutine of a general curvefitting program, the fit of the measured CD amplitudes was carried out stepwise. As starting procedure, with arbitrarily chosen K and w for fixed a = 2, the best values of A t p and A t p b were found. For different values of K , the fit with respect to A t p and A t p b was repeated until a value of K was found with the least variance in comparison to the foregoing runs. Starting with this value of K , a fit with respect to Atp, A t p b and K was done in an iterative manner (At is a nonlinear function of K ) . Repeating this iteration for different values of o,by the criterion of least variance, an initial value of o for an iterative fit with respect to Atp, Atpb, K , and o was found. Finally, a minimum of the variance with respect to all parameters Atp, Atpb, K , W , and a was reached. With the procedure described, one can be sure of having arrived a t an actual minimum. As a result of the calculations, the best fit of the experimental points was found for the following set of parameters:

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INTERACTION OF CU(I1) WITH NATIVE DNA

64 1

K = (116 f 36) mo1-I 1. a = (2.6 f 1.3) w =

(90 f 50)

The binding curve corresponding to these parameters is given in Fig. 8(b, full line), together with the experimental points. T o demonstrate the sensitivity of the model with respect to the size of binding sites, as well as to test the results given in the literature,22.26the best fit with a = 2 fixed will also be given. We found K = (115 f 26) mol-l 1. and w = (103 f 24). The corresponding binding curve [dotted line in Fig. 8(b)] in most of the region practically coincides with the curve first mentioned. From this and from a comparison of the variances of both fits (1.097 X lop3and 1.195 X respectively), it may be seen that the dependence on the parameter a is not very marked. The high values of the cooperativity parameter w confirm that the denaturing complex formation is a strongly cooperative process.

Scheme of Possible Reactions in the DNA-Cu2+ System As a result of the above equilibrium studies on DNA-Cu2+ complexes, the conditions (relative ligand concentration rt and temperature T )for the formation of different types of complexes have been established. These, together with some further data on complex dissociation, are summarized by the scheme of Fig. 10,where arrows in different directions indicate either an addition of ligand ions or a change of temperature or both. Thus, a nondenatured complex (DNA-Cu2+)n,t is formed a t low temperature T I on addition of a small amount of Cu2+ions to native DNA (horizontal arrow to the right rt = 0 rtl). Starting from this nondenatured complex, the formation of denatured complexes (DNA-Cu2+)denatcan be brought about either by further addition of Cu2+ions at temperature T1 (horizontal arrow rtl rt2) or by an increase in temperature (vertical arrow T I - T2). Alternatively, the state (rtl, Tz), a t which a denatured complex exists may be reached in two other ways: either by first heating the DNA in the submelting temperature range from T1 to T2 and then adding the ligand ions at Tz or (using a special experimental technique) by simultaneously adding the ligand ions and raising the temperature, according to the diagonal arrow from state (rt = 0, TI) to state ( r t l , T2). Complexes DNAdenat-Cuz+between heat-denatured DNA and Cu2+ions can be formed by first melting the DNA and then either adding the ligand ions at the high temperature T3, as indicated in the scheme, or adding the Cu2+ions after cooling of the heat-denatured D N A to temperature 7'1, as mentioned earlier. It is known from the literature2J8J9 that DNA that has been heat-denatured in the presence of Cu2+ions does not show any renaturation on

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t

r,,

0 R E L AT IV E

C 0 PPE R

rt2

C 0 NC E N T RAT I 0 N

Fig. 10. Scheme of possible reactions in the DNA-Cu2+ system, indicating various ways to reach different types of DNA-Cu2+ complexes. The complexes are characterized by the values of relative copper concentration rt and temperature T (0 < r l l < rt2 and 7'1 < T2 < 7'3 with T3 above the melting temperature of DNA). Broken-lined arrows to the left symbolize the dissociation of DNA-Cu2+ complexes by the addition of salt or EDTA (see text).

cooling, i.e., the hyperchromism resulting from denaturation is fully preserved. With regard to the scheme of reactions considered, this means that a renaturation of the denatured complex (DNA-Cu2+)denatcannot be brought about by just lowering the temperature according to a transition from state ( r t l ,T2) to state ( r t l , Tl). If, on the other hand, salt or EDTA is added a t ( r t l , T I )to the denatured and cooled complex, the complex becomes dissociated, and fully renatured DNA is obtained.lsJg Analogously, the renaturation of the denatured complexes a t states (rtl, 7'2) or (rt2, T I ) can also be induced by the addition of salt or EDTA, resulting in native DNA a t temperatures T2 or TI, respectively (symbolized in the scheme of Fig. 10 by broken-lined arrows to the left). The dissociation of the nondenatured complex ( DNA-CU~+),,~ can be reached in the same way, as shown in Fig. 11: salt is added up to a final concentration of 0.5M NaCl to the complex (DNA-CU~+),,~ in 0.01M NaCl ( r t = 1). By this procedure the decrease of the positive CD band, which is characteristic of the complex, is reversed, and the CD spectrum arising in high ionic strength is close to that of the initial native DNA. The slightly lower CD amplitudes can be explained by the dependence of the CD spectrum on salt con~entration.~G

INTERACTION OF CU(I1) WITH NATIVE DNA

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A Inml Fig. 11. Dissociation of the nondenatured complex (DNA-Cu2+),,, achieved by raising the ionic strength as revealed by CD spectra. Conditions: erythrocyte DNA, t = 25"C, CDNA = 1.0 X lO-*M (P). Spectrum 0, native DNA in 0.01M NaCI; spectrum 1, DNA-Cu2+ complex with rt = 1 in 0.01M NaC1; spectrum 2, after addition of up to 0.5M NaCl to the complex.

Kinetics From the scheme of Fig. 10, it becomes clear that by varying both ligand concentration and temperature, there exist several possibilities of forming different DNA-Cu2+ complexes. In order to gain an insight into the reaction mechanisms involved and to characterize further both nondenaturing and denaturing complex formation, we performed kinetic experiments on this system. The question to be answered was whether different kinetics are characteristic of the different ways of reaching a denatured complex (DNA-Cu2+)denat. Thus we applied several kinetic techniques, namely, manual mixing, stopped-flow, temperature-jump, and combined stopped-flow/temperat~re-jump.~3 In order to study the influence of the ligands on DNA denaturation kinetics, the results had to be related to data on the kinetics of thermal denaturation of DNA. We were especially interested in comparing the kinetics of thermal denaturation of the nondenatured complex (DNAC U ~ + ) with , , ~ ~that of DNA a t acid pH, because a certain analogy can be drawn: in both protonated DNA,56-61 symbolized by (DNA-H+)nat, and the nondenatured complex (DNA-Cu2+),at considerable conformational

FORSTER ET AL.

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changes are known to exist, and melting in both cases occurs with increased cooperativity a t the same strongly lowered t e r n p e r a t u r e ~ . ~ . ~ 8 ~ ~ 9 , ~ ~

Mixing Experiments

Nondenatured Complex (DNA-Cu2+),,t Studying the kinetics of formation of the nondenatured complex (DNA-Cu2+Inat,it could be expected that the primary bimolecular binding steps between the Cu2+ions and DNA are too fast to be detected by the stopped-flow technique. On the other hand, because of the CD results, it was necessary to test whether the strong conformational changes induced by the binding would cause some specific kinetic effect. Stopped-flow experiments on the nondenaturing complex formation showed that at the concentrations necessary to obtain a large enough absorbance change suitable for optical detection, the reaction was too fast to be resolved.63 (Typical example: CDNA = 10-4M ( P ) ,rt = 1,t = 25OC, solvent 0.01M KCl, wavelength X = 295 nm, deadtime of the instrument 2 msec.) The same was true for the dissociation reaction of the complex induced by a salt jump. (In the corresponding stopped-flow experiments, the salt concentration was increased from 0.01 to 0.2 or 1M KC1, respec0 l f

100 I

t rsi

200 1

31' C 0.1

25'C 27OC

34O C

37oc

-

Fig. 12. Kinetics of denaturing complex formation of erythrocyte DNA with Cu*+ ions a t different temperatures. Stopped-flow experiments, jumps rti = 0 rtf = 1.6. Conditions: 0.01M KCI, C D ~ = A 1.8 X 10-4M ( P ) ,X = 275 nm. Semilogarithmic plot of the normalized absorbance changes vs time, where At is the absorbance at time t, and A0 and A, are the initial and final absorbances, respectively.

INTERACTION OF CU(I1) WITH NATIVE DNA

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tively.) However, before concluding that the binding steps and conformational changes in nondenaturing complex formation and complex dissociation cannot, in principle, be detected by flow methods, it would be necessary to perform stopped-flow experiments with detection by CD.

Denatured Complex (DNA-Cu2+)denat In the mixing experiments at a given temperature, the relative ligand concentration rt was increased from an initial value rti to a final value rtf by the addition of Cu2+ ions to DNA. In the following discussion the extent of denaturing complex formation a t equilibrium corresponding to a given value of rt will be expressed by the ratio hlh,,, of actual hyperchromism h(hi or h f , respectively) to maximal hyperchromism h,,, a t saturation. The kinetic characteristics of the denaturing complex formation were studied as a function of temperature, as well as of the values of rti and rtf.43 Similar experiments on "copper( 11)-induced DNA denaturation kinetics," using a slow mixing technique, have already been r e p ~ r t e d . ~ ~ , ~ ~ However, only a qualitative comparison with our data is possible, because in the work cited only Cu2+equilibrium concentrations are given and not rt values or related parameters. The equilibrium concentrations were calculated on the basis of binding parameters, which according to our results cannot be true. Typical kinetic curves obtained in stopped-flow experiments on denaturing complex formation at different temperatures are shown in Fig. 12. Here, for jumps rti = 0 rtf = 1.6, the normalized absorbance changes at 275 nm are plotted semilogarithmically vs time. An analysis of the kinetic curves according to second-order kinetics was not ~uccessfu1,~~ from which

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Fig. 13. Evaluation of kinetic parameters by standard replotting procedure. Stopped-flow experiment from Fig. 12 a t 37°C. Three relaxation processes can be resolved. The corresponding relaxation times rt and amplitudes 0,(i = 1-3), determined from the slopes and intercepts of regression lines through the linear parts of the semilogarithmic plots, are as follows: T I = 86 f 2 sec, 01 = 0.18; ~2 = 11.1 f 0.5 sec, 0 2 = 0.28; r3 = 2.7 f 0.1 sec, 0 3 = 0.46.

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we conclude that an initial bimolecular binding step is too fast to be resolved (as already mentioned for the nondenaturing complex formation). In accordance with data from the literature on DNA denaturation kinetic~,~8?66,67 the curves could be described by a sum of exponentials with relaxation times ~i and amplitudes Pi. This is in agreement with the findings of the work cited a b ~ v ewhere , ~ ~in~addition ~ ~ to a “terminal” relaxat,ion process, a second, faster one was observed. Though our manual mixing experiments also showed only two relaxation processes, three such components could be resolved in the stopped-flow experiments shown in Fig. 12. They probably indicate that conformational changes connected with the denaturation process occur on complex formation. The standard replotting procedure38 for evaluating the kinetic parameters 7;and P; (i = 1-3) is demonstrated in Fig. 13 for one of the curves of Fig. 12. The temperature dependence of these kinetic parameters is shown in Fig. 14. The lower part [Fig. 14(b)],besides showing the amplitudes p;, gives their sum, indicating which part of the total optical effect is described by the three relaxation processes. Analogously, the upper part [Fig. 14(a)],

400 a)

Ti C s l

3 00

200

\* 100

0

1

PI 0.5

0 25

28

31

34

37

co

tpcl

Fig. 14. Temperature dependence of the kinetic parameters of the denaturing complex formation. Stopped-flow experiments as in Fig. 12, mean values of 3-6 experiments a t each temperature. (a) Relaxation times T~ and integral relaxation time i (see text). (b) Amplitudes @,and sum of amplitudes ZL@, (i = 1-3).

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besides showing the relaxation times ~ i presents , an “integral” relaxation time66 7, defined by 7 = Z i pi 71. As may be seen from Fig. 14, the largest relaxation time 71, in contrast to the shorter ones, is strongly dependent on temperature. I t decreases with increasing temperature, i.e., with the increasing extent of denaturing complex formation. At hflh,,, > 0.9, which under the conditions chosen is true for t > 37”C, a break in the character of the temperature dependence of 7 1 occurs (Fig. 14). In fact, it seems th a t the relaxation process, which corresponds to 7 1 , is no longer present a t these high values of hflh,,,. Obviously, a change in the mechanism of the reaction takes place (a similar transition dependence of the terminal relaxation time has been observed p r e v i ~ u s l y ~ ~An ) . overall acceleration of the reaction is also indicated by the amplitudes pi. From Fig. 14(b) it may be seen that a t 40°C a considerable part of the reaction is faster than the relaxation processes corresponding to 7 2 and 73. In the region of low denaturing complex formation (hflh,,, < 0.2), on the other hand, rather large values of 7 1 are found [Fig. 14(a)]. This is true not only for experiments a t low temperatures, corresponding to jumps (rt = 0, T I ) (rt2, T I )in the scheme of Fig. 10, but has also been observed a t higher temperatures ( t = 30-4OOC) for small enough values of r t , corresponding to jumps (rt = O,T2) ( r t l , T2). From this it is concluded th a t the kinetic parameters should be estimated with respect to the extent of denaturing complex formation, irrespective of the way by which it is brought about.43 In our opinion it is most likely that the denatured complexes (DNA-Cu2+)denatthat are indicated in the scheme of Fig. 10 a t states ( r t l , T2) and ( r t 2 , TI) are identical. Concerning the amplitudes pi shown in Fig. 14, we observe that for temperatures t < 3OoC,a large part of the total reaction is not resolved in time and is not enclosed in the three relaxation processes. This is due to the fact t ha t a t the wavelength of 275 nm, the sum of both the nondenaturing and the denaturing complex formation is detected. A t low temperatures the relative contribution of the nondenaturing complex formation to the observed overall reaction is large, resulting in a large “instantaneous” optical effect. In another kind of manual mixing experiment, Cu2+ions were added a t a given temperature to DNA-Cu2+ complexes with different initial ligand concentrations rti. A constant final value rtf was reached. Such experiments are shown in Fig. 15 (jumps rti rrf = 1.2 with rti varied). The fast relaxation process corresponding to 7 3 , which was detected in the stopped-flow experiments mentioned above, is not resolved in time. As indicated by the constant slopes of the terminal parts of the kinetic curves in semilogarithmic presentation, the largest relaxation time 71 obviously does not depend on the initial state rti. T h e amplitude PI, on the other hand, increases with decreasing “jump size” and becomes maximal for small jumps. [In the limit it is close to unity, as may be seen from the intercept +

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I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

0

0 0.0

nc

0.

0 0.L

77 %

6

-9

90 %

n

-

Fig. 15. Kinetics of denaturing complex formation of erythrocyte DNA with Cu2+ions a t rtf = 1.2, with initial relative ligand constant temperature. Manual mixing, jumps rt, concentration rti varied. Conditions: 0.01M KC1, t = 40.2OC, CDNA = 2.3 X 10-4M (PI, X = 275 nm. Semilogarithmic plots of the normalized absorbance changes vs time. Jumps rtr rtf are characterized by the corresponding extents (percentage) of complex formation h,/h,,, hf/h,,, (see text). Two relaxation processes are resolved for large enough jumps.

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INTERACTION OF CU(I1) WITH NATIVE DNA

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of the kinetic curve with the ordinate (Fig. 15, lower part)]. Because of this property of P I , the integral relaxation time 7 increases with decreasing jump ~ize.~3 For other mixing experiments, in which different amounts of Cu2+ions rtf with rtf were added to DNA a t a given temperature (jumps rti = 0 varied), the integral relaxation time 7 showed a maximum around hflh,,, = 0.5 (studied for t = 37 and 4OoC43). This result should be compared with the experiments of Fig. 14, where for a given value of rtf the temperature was varied. Here, too, we observe a maximum of 7 when the extent of denaturing complex formation is about hf/hmax = 0.5, which in Fig. 14 is true for t = 31°C. This similarity again confirms the significance of the parameter hf/hma,in the kinetic characterization of the denaturing complex formation, irrespective of the combination ( r t , T ) chosen to bring it about. The kinetics of the renaturation of the denatured complex (DNACU2+)denatinduced by raising the ionic strength after cooling has been studied extensively by several a ~ t h o r s . ~ ~ We - ~ Ohoped to obtain further information on the mechanism of the denaturing complex formation from "time-dependent" renaturation experiments using multimixing stoppedflow equipment. By using this technique at elevated temperature T2 after a first mixing of DNA with Cu2+ions (jump rti = 0 r t f ) ,which resulted in the complex (DNA-Cu2+)denat,a second mixing of this complex with a KCl or EDTA solution at a predetermined time initiated the renaturation reaction. The kinetics of renaturation was followed optically. The time t* between the two mixing events, i.e., the time given for the denaturing complex formation to proceed, was varied. I t was hoped that from the dependence of the renaturation kinetics on this time t*, some conclusions could be drawn concerning the nature of the relaxation processes involved in the denaturing complex formation. We observed first-order renaturation kinetics in our time-dependent salt-jump renaturation experiments. (Experimental conditions: CDNA = 0.5 X 10-4M ( P ) ,solvent 0.01M KC1, rt = 2, t 2 = 55"C, X = 275 nm, time t* varied between 10 min and 0.2 sec, salt-jumps to 0.2M KC1.) This is in contrast to studies of ordinary DNA renaturation kinetics, which start from heat-denatured single-stranded DNA and show second-order kinetics, a t least in the early stages of the process, because of rate-limiting nucleation e ~ e n t s . ~ O ,The ~ l difference in reaction order shows that no strand separation occurs during the denaturing complex formation of DNA with Cu2+ ions. However, under the experimental conditions chosen, no significant dependence o f the renaturation kinetics on t* could be established. Nevertheless, further investigations with a more appropriate ratio between the time constants of the denaturing complex formation and the available time range for t* might confirm the use of the approach described.

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Temperature- Jump Experiments

Thermal Denaturation Kinetics o f Protonated D N A For the purpose of comparison with the kinetics of the thermal denaturation of the nondenatured complex (DNA-CU~+),,~,we studied the pH dependence of the kinetics of the thermal denaturation of DNA at low ionic ~trength.~~,~3 In Fig. 16 the integral relaxation times 7 obtained in a series of temperature-jump experiments with s.chrysomallus DNA at different pH values are presented as a function of the jump size. Temperature jumps were made from a temperature a t the beginning of the melting transition to a temperature within the melting transition, resulting in a certain final degree of denaturation (1 - O f ) . A t pH 6 the DNA denaturation kinetic? can be described by one single relaxation time T , which in this case coincides with the integral relaxation time 7 given in Fig. 16. It obviously does not depend on the value of (1 - O f ) . By lowering the pH value the melting transition of DNA is shifted to lower temperatures.62 To be consistent, we compared those temperature jump experiments which resulted in the same final degree of denaturation (1 - O f ) . Figure 16 shows that a t pH 3.95 somewhat larger values of 7 (and of the relaxation time 7)are found than a t pH 6. A slight dependence of 7 on (1 - 0,) is observed. On larger DNA protonation (pH I 3.8), the kinetic curves cannot be further described by only one relaxation time. Two relaxation processes are detected, with the larger relaxation time T I strongly dependent on the jump size (shown below). The values of 71, as well as those of the corresponding amplitude 01, decrease with increasing jump size. At pH 3.7 the relaxation time 7 9 of the faster process decreases from about 10 sec a t 0.4 I (1 - Of I 0.6 to about 2 secat the end of the tran~ition.~3For large pHz3.7 tm=3C pHz3.8 tm=365’ pH-3.95 tm=LWpH=6 tmz79’C

0 0

0.5

1

05

1 0

05

0 1

0.5

(1- g1

Fig. 16. Kinetics of thermal denaturation of DNA at different p H values. Integral relaxation time ?as a function of jump size for temperature jumps from the beginning of the melting transition (see text). Conditions: S. chrysornallus DNA, pH adjusted by mixing solutions of Hap04 in KCI with KOH, 0.01M K+ a t pH 6 and 0.02M K + otherwise, C D N A = 1.9 X 10-4M ( P ) . Values of pH and of melting temperature t , are indicated. Abscissa: final degree of denaturation (1 - of).

INTERACTION OF CU(I1) WITH NATIVE DNA

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jumps this fast relaxation process predominates [& increases from about 0.4 a t (1- 0,) = 0.4 to 1.0 at (1- 0,) L 0.951. T h e resulting integral relaxation time ? in the case of maximal protonation (pH 3.7, Fig. 16) depends very strongly on the final degree of denaturation. Thermal Denaturation Kinetics of the Nondenatured Complex (DNA-CU 2+)nat In analogy to the above experiments with protonated DNA a t different pHs, temperature-jump experiments with the complex (DNA-Cu2+),,t were done for different relative ligand concentration^.^^ By increasing the value of rt, the melting transition of the complex is shifted to lower temperatures,ls,lg as is the case with DNA a t decreasing pH. The temperature-jump program was as described above. The kinetics was studied as a function of the final degree of denaturation (1 - B f ) , which has to be identified here with the extent of denaturing complex formation h fl hill,,. A t the smallest value o frt used in the experiments (rt = 0.35), the kinetic curves, as in the case of slightly acid pH, could be described by a single relaxation time of about 10 sec that was only slightly dependent on jump size. At larger relative ligand concentrations (rt 2 0.5),however, a specific kinetic effect was found: the kinetic curves were no longer of exponential character, but a lag in the rise of optical density following the temperature jump was observed.42 After this initial delay of the response, the kinetics again could be described by a sum of two exponentials. Altogether, a sigmoidal form of the kinetic curves resulted, as is shown in Fig. 17 for several experiments with rt = 1 (curves 1-4). For the purpose of comparison, Fig. 17 also shows a typical exponential kinetic curve taken from the temperature-jump experiments with protonated DNA (curve 5). Because of similar experimental conditions (jump temperatures are identical for curves 1and 5), the possibility that artifacts resulting from differences in technique have caused the different character of the kinetic curves can be excluded. The lag period observed was longest for small jump sizes with (G C)rich DNA a t Cu2+concentrations for which the melting temperature was lowered maximally. For rt = 1.4 (t, = 31.5”C), the lag period was as long as 20 sec. Figure 18 shows for different values o f r t the relaxation time 7 1 of the final relaxation process as a function of (1- O f ) . Values (1- O f ) > 1denote “large” temperature-jumps onto the plateau of the melting curve. The relaxation time 71 strongly depends on the jump size and reaches values t ha t are much larger than in the case of protonated DNA (Fig. 18), as a comparison of a series of experiments with similar melting temperatures shows. An integral relaxation time, by definition, cannot be determined for the sigmoidal kinetic curves. The temperature-jump experiments described above have been performed with the apparatus using microwave heating. The sigmoidal shape of the kinetic curves has also been observed in temperature-jump experiments with the nondenatured complex (DNA-CU~+),,~using the

+

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0.1 0

--4cc

-

20

ia

30

t [s]

Fig. 17. Typical kinetic curves obtained in temperature jumps with the nondenatured complex (DNA-Cu2+),,t and with protonated DNA. Absorbance changes AAt = At - A0 vs time t , final absorbance changes AA, for the different experiments are indicated. Conditions: S. chrysornallus DNA, CDNA = 1.9 X 10-4M ( P ) . Curves 1-4: (DNA-Cu2+),,,, 0.01M KCI, rt = 1.0, h = 275 nm; curve 5: (DNA-H+),,t, 0.02M K+, pH 3.8, X = 270 nm. Jump temperatures for curves 1-5: t l = 30°C in all cases; tz = 35,36,37,38, and 35"C, respectively.

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stopped-flow method bumps ( r t l , T I ) ( r t l , T2);see scheme of Fig. 101. These experiments were performed by mixing the complex a t temperature T I with solvent a t temperature T3 to reach the final temperature T2.fi3 If, on the other hand, DNA of temperature T I was mixed with a Cu2+ solution of temperature T3, thus performing a simultaneous mixing and temperature-jump experiment [the diagonal arrow (rt = 0, T I ) ( r t l , 7'2) in the scheme of Fig. lo], the kinetic curves were of exponential character. No qualitative difference of the kinetics could be found when compared with the stopped-flow experiments on the denaturing complex formation presented earlier (Figs. 1 2 and 13),which corresponded to a transition (rt = 0, T2) (Ql, T2). From this we conclude that the observed initial inhibition of the denaturing complex formation after temperature-jump must be connected with some special structures present in the nondenatured complex (DNAC U ~ +prior ) ~to~the ~ heating.63

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INTERACTION OF CU(I1) WITH NATIVE DNA r,=lL

rt=l.O

tm=31.5’

tm=35’

rt=07

0.5

1

0

05

rt=05

rt=035 tm=5C

t,.43’

7

1

0

tm=39’

653

---I--1

0

05

1

0

05

1

0

05

1

Cl-gl

Fig. 18. Kinetics of thermal denaturation of the nondenatured complex (DNA-CuZf),,t a t different values of rt. Relaxation time T I of the final relaxation process as a function of the jump size (see text). Conditions: S. chrysornallus DNA, 0.01M KCI, CDNA = 1.9 X 10-4M (PI. Values of 71 for the experiments with protonated DNA at pH 3.7 (cf. Fig. 16) are included (open circles).

From the stopped-flow experiments which could not resolve the nondenaturing complex formation, it should not be concluded without further investigation that the special structures in the nondenatured complex (DNA-CU~+),,~are formed “immediately” after mixing. In our opinion combined stopped-flowhemperature-jump experiments, in which the time t** between the mixing and the temperature-jump should be varied, could give information about the kinetics of formation of these structures. In the experiments presented, only the limits t** = 0 and t** = m were studied. The minimal value of the time t** after which sigmoidal curves in the temperature-jump kinetics appear should be related in some way to the “time constant” of formation of these structures.

A NEW BINDING MODEL On the basis of the experimental results presented, a clearcut distinction has to be made between two qualitatively different types of complex formation of native DNA with Cu2+ions a t low ionic strength. This has obviously been overlooked by previous authors who demonstrated a n interaction of DNA with the ligands at room t e m p e r a t ~ r e . ~ ~ - ~ ~ , ~ ~ If one tries to formulate a more general model of the interaction of Cu2+ ions and other divalent transition metal ions such as Cd2+,Zn2+,or Mn2+

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with DNA, the following additional experimental facts must be considered. 1. Ir measurements, performed a t conditions under which only the nondenaturing complex formation takes place, have shown that the bases guanine and cytosine are involved in the binding of Cu2+ ions to native DNA.23,25 In the experiments cited, the complexes were formed in 0.006M NaC104, and the relative ligand concentrations were rt = 0.1 for S. chrysomallus DNA and rt = 0.016 and 0.1 for Sarcina maxima DNA (erroneous values of rt were given in Ref. 25). 2. Changes in the CD spectrum of native DNA on complex formation with Cd2+and Zn2+ ions5 are qualitatively very similar to those observed for the nondenatured complex (DNA-CU~+),,~.Less pronounced, but in the same direction, are the changes induced by Mn2+, Co2+,and Ni2+ ions. 3. In the CD spectrum of protonated DNA a new band is present, which is assumed to be characteristic of an anti-syn conformational transition of guanosine about the glycosidic bond.59,60,72This CD band disappears on addition of Zn2+ ions t o protonated DNA.5 4. For the nondenatured DNA-Cu2+ complex at high ionic strength and a t very low relative ligand concentrations (0.1M NaC104, rt = 0.03), a selective thermal stabilization of the G-C pairs is found.24 5. The melting temperature of DNA can be lowered by the addition of divalent transition metal ions only in the cases of Cu2+and Cd2+ions2 On complexing with Zn2+ ions, the melting temperature of DNA initially increases, while it decreases a t larger values of rt, nearly reaching the melting temperature of DNA without l i g a n d ~ . ~ ? ~ ~ 6. On cooling, the heat-denatured DNA-Zn2+ complex renatures completely, whereas no renaturation can be detected on cooling the heat-denatured DNA-Cd2+ or DNA-Cu2+ complexes.2 Complete renaturation in the two latter cases can be induced by increasing the ionic strength2 or by addition of EDTA (shown for the DNA-Cu2+ c ~ m p l e x ' ~ ) . 7. The affinity of the ligands for the different bases has been estimated from studies on the influence of divalent metal ions on the conformation of polyribonucleotides."O The ions may be classified into two groups, especially in the case of poly(C): a t room temperature, both Cu2+and Cd2+ ions, because of their strong affinity for cytosine, produce the random-coil form from the double-helical structure of poly(C), as well as from its single-stranded helical form.3o The same conformational changes can be observed on heating. Zn2+ ions a t the same concentration, on the other hand, do not significantly affect the conformation of poly(C), but much larger ligand concentrations are necessary to produce the random-coil form. With Ni2+, Mn2+,or Co2+ ions, no base binding in poly(C) is observed.3o The literature suggests mainly two models of the structure of DNA-Cu2+ complexes to explain some of the experimental findings mentioned. In order to explain the ir results, Zimmer et al.25postulated a complex in which a Cu2+ion is bound to a G-C pair between the bases guanine and cytosine.

INTERACTION OF CU(I1) WITH NATIVE DNA

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Coordinate bonds are assumed to form in this interstrand complex between the ligand ion and atoms “7) and 0 of c(6)of guanine, as well as N(3)and 0 of C(2)of cytosine, after a transition of deoxyguanosine from the anti into the s y n conformation. In this way a simultaneous binding of one Cu2+ion to the most probable binding sites in both guanine and cytosine would explain the ir results mentioned in point 1. The crosslink properties of the postulated structure are thought to be correlated with the renaturation behavior mentioned in point 6. In our opinion this model cannot be true because of the following reasons. The absence of an additional CD band in the CD spectrum of the complex (DNA-Cu2+),at (Figs. 1 and 2) contradicts the assumed change of the guanosine conformation from anti to syn. Especially for the (G C)-rich S. chrysomallus DNA, we should expect evidence of this CD band near 260 nm, which according to point 3 is characteristic of an anti-syn transition, if the Cu2+interstrand complex a t G C pairs actually forms with a sufficient binding constant, as has been suggested. It is not only the lack of this band in the CD spectrum of the complex (DNA-Cu2+)natthat favors the assumption of the anti conformation of guanosine in the complex. Because of the findings in points 2 and 3, one has to expect that the addition of Cu2+ ions to protonated DNA would also result in the disappearance of the characteristic CD band mentioned, as is the case with Zn2+ions. Therefore, a binding model with guanosine in the s y n conformation seems unlikely. Further difficulties arise if the interstrand complex is considered as the preferred structure in the denatured complex (DNA-Cu2+)denat,as has been done.3 In this case the well-known properties of d e n a t u r a t i ~ n ’ ~ cannot J~.~~ be explained, because a modified, even more rigid, double-stranded structure with the hydrogen bonds between guanine and cytosine replaced by coordination of a Cu2+ion should be expected for the complex. Other problems concerning the mechanism of renaturation exist with the interstrand complex, as has been pointed out.26 An alternative model has been favored by Daune e t al.24,26in order to explain the experimental results obtained on the DNA-Cu2+ interaction a t high ionic strength (point 4 above) and to account for the crosslink properties responsible for the renaturation behavior (point 6 above). According to this model, a “charge-transfer-type” complex with both guanines as donors, the Cu2+ as acceptor, and the metal ion intercalated between the two G-C pairs is formed in native DNA, even a t high ionic strength, a t the G-C pairs with sequence dGpG. From model-building studies for this type of intrastrand complex, it was originally postulated that a coordinate binding of the Cu2+ions takes place between atoms N(7) and 0 of c(6)of both guanines in the same strand. By a redistribution of charges in the G-C pairs, the cytosine bases are thought to be influenced by the binding too, and an increase in the stacking energy of the G-C pairs is assumed to occur.26 Nevertheless, it is difficult to understand how it would be possible to get the interstrand crosslinking necessary to explain the renaturation behavior from a mainly intrastrand complex structure.

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If nitrogen atoms N(7)of the guanines really play a predominant role in this complex, as has been supposed, then it is not clear how an “intercalation” of the ligand ions between the base pairs inside the double helix might occur. Summarizing, both complex structures discussed turn out to have serious shortcomings if all the known experimental facts are considered. In order to arrive at a more consistent model of the complex formation between Cu2+ions and native DNA at low ionic strength, we suggest the following modes of interaction: Several types of complexes are formed simultaneously on nondenaturing complex formation, some stabilizing and some destabilizing the DNA secondary structure. The complex structure responsible for the renaturation behavior is coupled with sites of special sequences of G-C pairs. The ligand ion is bound between the base pairs inside the double helix. Coordinative bonds are formed between the metal ion and electronegative atoms in guanine and cytosine bases simultaneously, thus accounting for the ir results and the crosslink properties. The syn conformation of guanosine does not occur in the binding process. For the denaturing complex formation of DNA with Cu2+ ions, the binding tendency of the ligands to cytosine is decisive. In more detail, the following different binding sites a t the DNA bases become occupied by Cu2+ ions during nondenaturing complex formation: a. Mixed chelates are formed with atoms N(7) of purine bases and the ~ ~ binding is sensitive corresponding phosphate groups as l i g a n d s . 3 ~ This to ionic strength and does not lead to a base-pair specificity. b. True chelates are formed with atoms N(7)and 0 of c(6)of guanine bases.3.25 This binding is G-C specific and results in a destabilization of the DNA secondary structure because of a loosening of one of the hydrogen bonds within the G-C pair. c. Cu2+ions become bound inside the double helix between two adjacent G C pairs. We assume a coordination of a Cu2+ion with atoms “3) of cytosine bases and atoms 0 of C(6)of guanine bases. Atoms N(7)of guanine bases, despite their strong donor properties, will not be involved directly in this mode of binding because of their unfavorable position. Model-building studies with CPK molecular models show that base pairs of sequence dGpC offer the most favorable geometry for an insertion of the metal ion and for its coordination with the atoms mentioned in an almost tetrahedral geometry. This is illustrated in the upper part of Fig. 19,where a view is given down the helix axis of the B-form DNA double helix with two adjacent G-C pairs of sequence dGpC. The position of equal distance from the four atoms N(3)and 0 of C(s)is indicated by a circle near the helix axis. The distorted tetrahedral geometry with respect to this potential center of coordination is shown separately. Furthermore, for all three possible sequences of two G-C pairs, the relative position of the lines con-

INTERACTION OF CU(I1) WITH NATIVE DNA

657

Fig. 19. Geometry of potential binding site of Cu2+ ions forming an interstrand complex at adjacent G.C pairs. Upper part: view down the helix axis of the B-form DNA double helix with base pairs of sequence dGpC (shown schematically). The four atoms N(3)of cytosine and 0 of C(6)of guanine bases assumed to be involved in the binding are indicated by dots, while the position of equal distance from these atoms is indicated by the circle near the helix axis (cross). The distorted tetrahedral geometry with respect to this potential center of coordination is drawn separately. For all three different sequences of two G C pairs, the projections of the lines connecting the mentioned atoms within each G C pair are shown (GpC, upper part; CpG and GpG, lower part).

necting atoms N(3) of cytosine and 0 of C(6)of guanine is indicated schematically. It can be seen that in the case of sequence GpC, the angle between these lines is nearly rectangular, while for the two other sequences, CpG and GpG, it clearly deviates much more from this value characteristic of a tetrahedron (lower part of Fig. 19). For the sequence GpC the angle mentioned becomes 90" by a slight local unwinding, i.e., by a decrease of the winding angle between the base pairs from 36" to about 29", and the geometry becomes more tetrahedral. Along with this effect the distance between the two base pairs increases by the unwinding, thus facilitating an insertion of the metal ion inside the double helix. These arguments lead us to postulate that during nondenaturing complex formation an interstrand complex with crosslink properties as shown in Fig. 19 will form a t sequences dGpC with a high association constant. The CD results reveal marked conformational changes of DNA on nondenaturing complex formation (DNA-CU~+),,~. While the specific contributions of the above three types of Cu2+ ion binding to the observed overall CD signal are not known, an interpretation of the CD changes in terms of a conformational transition to a C-like form will probably be a rather crude approximation. Local sequence-dependent effects on con-

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formation must be envisaged, and studies with synthetic DNAs of different base sequence would be necessary to clarify the situation. The nondenaturing complex formation described in this paper for the interaction of native DNA with Cu2+ions obviously takes place with Cd2+ and Zn2+, and probably Mn2+ ions too. This is concluded from the CD changes mentioned in point 2 above, as well as from the thermal denaturation and renaturation properties of the complexes mentioned in points 5 and 6. From these experimental findings a general order of increasing relative affinity of the ligands for base sites compared to phosphate sites in DNA has been e s t a b l i ~ h e d ~ ,Ni2+, ~ : Co2+ < Mn2+ < Zn2+ < Cd2+ < cu2+. In our opinion it is not only the overall relative affinity of the ligand ions for base and phosphate sites that is important to the effects of binding on the conformation and stability of the secondary structure of DNA but also the affinity of these ions for the individual heterocyclic bases, which may be decisive in some cases. We suppose that this is true for the denaturing complex formation (DNA-Cu2+)denat,for which we suggest the following m e c h a n i ~ m . ~ 3 9Due ~ ~ to - ~their ~ strong binding tendency to cytosine bases, the Cu2+ions bind to atoms N(3)of cytosines when these bases come to the outside of the double helix by statistical fluctuation^.^^ This binding is sufficiently strong to prevent the bases from returning to their former positions. Thus local denaturation a t G-C pairs is brought about. This mechanism is capable of conveniently explaining several of our main experimental results and allows us to make some predictions: As mentioned earlier, by nondenaturing complex formation (DNAC U ~ + )within , ~ ~ G-C pairs that do not belong to a sequence dGpC, a hydrogen bond is weakened because of the chelate formation of a Cu2+ion with guanine. This should give rise to an enhanced fluctuational opening of the DNA double-helical structure and result in denaturing complex formation. On this basis one can understand the denaturing action of Cu2+ ions a t sufficiently high ligand concentrations, even a t the low temperatures characterized by state (rt2, 7'1) in the scheme of Fig. 10. By raising the temperature, on the other hand, the structural fluctuations in DNA or in the nondenatured complex (DNA-CU~+),,~ are also enhanced and denaturing complex formation becomes possible a t state (rtl, 7'21, as indicated in Fig. 10. The above mechanism is in agreement with the observed temperature dependence of the half-transition points of the spectrophotometric titration curves (Fig. 9). It is also in line with the temperature dependence of the largest relaxation time T~ in the kinetics of denaturing complex formation [Fig. 14(a)],if it is assumed that 7 1 reflects the dynamics of the statistical helix-opening process, which would be rate limiting here. In the mixing experiments, the finding that for a given final extent of denaturing complex formation in the two cases characterized by the final states (rt2, TI) and ( r t l , T2),the kinetic parameters are largly independent of the combination (rt, T ) , points to a common mechanism, as suggested.

INTERACTION OF CU(I1) WITH NATIVE D N A

659

Our predictions are as follows: Qualitatively, the same kind of denaturing action on binding to DNA as found for Cu2+ ions has to be expected for Cd2+ions because of their sufficiently strong binding tendency to cytosine bases. This we conclude from the binding studies with polynucleotides mentioned in point 7. By ir measurements an additional binding of the ligands to cytosines should be detectable on denaturing complex formation. (So far, ir measurements have been performed only at the conditions of nondenaturing complex formation.) Furthermore, it should be possible to obtain from one and the same ir spectra information on the degree of ligand binding to cytosines (ir band a t 1550 cm-l; Ref. 25) as a function of the extent of denaturing complex formation (estimated from the absorbance ratio a t wavenumbers 1665 and 1685 cm-’; Ref. 40). The difference in binding tendencies of the divalent transition metal ions to cytosine bases offers, in our opinion, a convenient explanation for the different renaturation properties of the denatured complexes of Cu2+,Cd2+, and Zn2+ ions with DNA (point 6). Because of the lack of strong ligand binding in the heat-denatured DNA-Zn2+ complex that has been cooled, the cytosines are not prevented from base pairing with guanines. The DNA strands are held in register by the interstrand crosslink complexes a t sequences dGpC, and complete DNA renaturation takes place, since the other types of Zn2+ ion binding in the denatured state are overcome by the helix-stabilizing forces at low temperature. In the denatured DNA-Cu2+ and DNA-Cd2+ complexes, on the other hand, even after cooling to low temperatures, the ligand ions remain bound to cytosines, thus preventing renaturation. Only after additional weakening of the binding, either by increasing the ionic strength or by means of a strong chelating agent, can the ligand ions dissociate from their binding sites and renaturation of the DNA proceed. Finally, concerning the thermal denaturation kinetics of the nondenatured complex (DNA-CU~+),,~, we explain the observed lag in response to the perturbation with the interstrand crosslink structures existing in the complex prior to the heating. This means that we identify the previously mentioned “special structures” responsible for the initial inhibition of the denaturing complex formation with the crosslinks a t G-C pairs of sequence dGpC. This is in agreement with the G C dependence of the kinetic effect under consideration. From a kinetic point of view, it seems reasonable that these special interstrand crosslinks within the double helix are not formed “immediately” on nondenaturing complex formation, as argued above in the discussion of time t**. In order to explain the lag period, we assume that a certain number of crosslinks have to be broken following the temperature jump before the denaturation via cytosines (followed by local unwinding and loop formation as main steps of denaturing complex formation) proceeds to such a degree that it can be detected optically. In summary, it seems to us that by means of the binding model we have

+

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FORSTER ET AL.

formulated above, all of the known experimental facts on the interaction of Cu2+ ions with DNA can be understood, a t least qualitatively, quite well. We wish to thank Dr. Ch. Zimmer for performing CD measurements and for many valuable discussions. We also thank Dr. L. A. Platonov for performing the microcalorimetric measurements.

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Received February 10,1978 Accepted July 1,1978