Studies on the binding of actinomycin D to DNA and DNA model polymers

J. 1Mol. Biol. (1970) 49, 319-342

Studies on the Binding of Actinomycin D to DNA and DNA Model Polymers R. D.

WELLS

AND

J. E. LARSON

Departmelzt of Biochemistry University of Wisconsin, Madison, Wk. 53706, U.S.A. (Received August 1969, and in revised form 21 November 1969) The ability of 17 different DNA’s to bind actinomycin D was studied by a variety of techniques including equilibrium dialysis, in. vitro transcription and analytical buoyant density centrifugation. The major conclusions are as follows: (1) The presence of deoxyguanylic acid in a DNA is not necessary for complex formation. Poly d1 binds approximately one-quarter as much AMt as DNA’s which contain 50% G + C; the equilibrium constant for the poly dI-AM complex is as large aa that observed for 50 y0 G + C DNA%. (2) The presence of deoxyguanylic acid in a DNA is not sufficient for complex formation. Poly d(A-T-C) .poly d(G-A-T), which contains 33% G + C, binds little or no AM as judged by five different techniques. (3) A marked nucleotide sequence preference exists for the binding reaction. When comparing sequence isomeric DNA’s, the poly d(pur,-pyr,) * poly d(pur,-pyr,) isomer binds more AM and binds AM more tightly than does the poly d pur * poly d pyr isomer. (4) The guanine-containing DNA’s tested, with the exception of poly d(A-T-C) * poly d(G-A-T), bind AM and are : Micrococcus Zuteus DNA, salmon sperm DNA, poly d(G-C) . poly d(G-C), poly dG * poly dC, poly d(T-G) epoly d(C-A), poly d(T-C) +poly d(G-A), poly d(T-T-G) * poly d(C-A-A), poly d(T-T-C) . poly d(G-A-A) and poly d(T-A-C) * poly d(G-T-A). (5) The DNA’s which are devoid of deoxyguanine, with the exception of poly d1, do not bind AM and are : poly d(A-T) . poly d(A-T), poly dA * poly dT, poly d1. poly d0 and poly d(I-C) . poly d(I-C). The results are discussed in relation to two models for the AM-DNA complex, the hydrogen-bonded, “outside-binding” model of Hamilton, Fuller & Reich (1963) and the intercalation model of Miiller & &others (1968). The data are not consistent with the hydrogen-bonded model.

1. Introduction acid to bind actinomycin is a sensitive indicator of polynucleotide configuration. Double-stranded DNA binds the antibiotic (for a review see Reich & Goldberg, 1964), whereas double-stranded RNA binds only poorly, if at all (Haselkorn, 1964). That double-stranded RNA has a slightly different configuration than double-stranded DNA has been shown by X-ray diffraction (Arnott, Wilkins, Fuller & Langridge, 1967). In addition, a hybrid polymer containing one-strand DNA and the complementary strand RNA binds little, or no, AMT. Again, X-ray analysis has shown that this molecule possesses a slightly different configuration than bihelical DNA (Milman, Langridge & Chamberlin, 1967). Denatured DNA, or single-stranded DNA, binds only poorly. Thus, the studies described were undertaken to determine if the AM-binding ability of synthetic polydeoxyribonucleotides could be used as a The capacity

of a nucleic

t The abbreviations for the polynucleotides used in this study have been described previously (Wells, Jacob, Narang & Khorana, 1967a) but have been modified to conform to IUPAC oonventions; AM, actinomycin D. 21

319

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R. D. WELLS

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J. E. LARSON

probe for secondary structure as previously suggested. Other studies (summarized in Wells, Larson, Grant, Cantor & Shortle, manuscript in preparation) have already indicated that all the DNA’s do not have identical structures. A series of DNA model compounds containing defined repeating nucleotide sequences has recently been synthesized by a combination of chemical and enzymic techniques (Byrd, Ohtsuka, Moon & Khorana, 1965; Wells, Ohtsuka & Khorana, 1965; Wells, Jacob, Narang & Khorana, 1967 ; Wells, Biichi, KGssel, Ohtsuka & Khorana, 1967 ; Morgan & Wells, 1968). In addition, a variety of simple DNA polymers are made de novo by DNA polymerase (Schachman, Adler, Radding, Lehman & Kornberg, 1960; Radding, Josse & Kornberg, 1962; Inman & Baldwin, 1964; Grant, Harwood & Wells, 1968). The availability of these DNA models has made possible a variety of chemical, enzymic and physical studies (summarized in Wells et al., manuscript in preparation). In the present communication, the AM-binding ability of a number of different DNA’s is reported. Actinomyoin is possibly the best known and most thoroughly studied antibiotic which blocks DNA function. Using a variety of analogs of actinomycin, it has been possible to establish the critical groupings on the molecule for binding (Reich, Cerami $ Ward, 1967; Mtiller & Crothers, 1968). On the basis of kinetic studies with AM analogs, as well as other considerations, Miiller & Crothers (1968) made predictions concerning the structure of the complex and the mechanism of its formation. We have, in a sense, done the complementary experiments by studying a variety of analogs of DNA. Our results demonstrate that the presence of deoxyguanylic acid is neither necessary nor suiIicient for the binding reaction. These results are contrary ,to the predictions from previous studies (Reich & Goldberg, 1964). Also a sequence preference for the binding process is established. Predictions on the structure of the AM-DNA complex as well as on the apparent role of deoxyguanylic acid are made possible by these studies. A brief report on part of this work has been published (Wells, 1969).

2. Materials and Methods (a)

DNA preparations

The procedures used for the preparation of the DNA-like polymers have already been described. The polymers were the following: poly d(A-T) * poly d(A-T) (Schachman et al., 196O);polydA~polydT (Byrd&al., 1965;Morgan,Wells&Khorana, 1967);polyd(I-C)*poly d(I-C) (Grant et al., 1968); poly dI * poly dC, poly dG . poly dC (Inman & Baldwin, 1964) ; poly d(T-C).poly d(G-A) (Byrd et al., 1965; Wells et al., 1965); poly d(T-G) *poly d(C-A) (Wells et al., 1965); poly d(T-T-C) *poly d(G-A-A), poly d(T-T-G)*poly d(C-A-A), poly d(T-A-C) epoly d(G-T-A) and poly d(A-T-C) apoly d(G-A-T) (Wells, Jacob, Narang & Khorana, 1967). Poly d.A * poly dT, poly dG . poly dC and poly d1 * poly dC were separated into their constituent single-stranded polymers by preparative alkaline density-gradient centrifugation by the method of Wells & Blair (1967) using a fixed angle Ti-50 rotor. The single-stranded polymers were then recombined in the appropriate molar ratio to ensure an equal ratio of the two strands. The two repeating dinucleotide DNA’s and the four repeating trinucleotide DNA’s were isolated by the warm phenol method described previously as method (b) (Wells, Jacob, Narang & Khorana, 1967). Poly d(A-T) * poly d(A-T) and poly d(I-C) . poly d(I-C) were isolated by the chloroform-isoamyl alcohol procedure as previously described (Morgan & Wells, 1968), since these DNA’s are appreciably extracted into the phenol phase under the above conditions. Poly d(G-C) . poly d(G-C) was prepared using poly d(I-C) epoly d(I-C) as a template for the Micrococc~s Zuteus DNA polymerase (generously provided by S. J. Harwood) and will be the subject of a forthcoming communication (Grant & Wells, unpublished work).

BINDING

OF ACTINOMYCIN

D TO DNA

321

Each of the DNA polymers was analyzed for purity and composition by analytical buoyant-density centrifugation, both in the absence and in the presence of added marker DNA. By this method each sample was shown to be free (estimated at less than 3 to 5%) of any other contaminating polymer. The buoyant densities in cesium chloride solution of the DNA polymers used in this study are identical to those reported previously (Wells & Blair, 1967; Grant et al., 1968). It is estimated that the amount of the two strands of each of the DNA’s (poly d(A-T) * poly d(A-T) and poly d(I-C) * poly d(I-C) excluded) are equal (within + 4%) as judged by dual isotopic labeling experiments (see Wells, Jacob, Narang & Khorana, 1967). This does not necessarily mean, however, that the DNA’s are entirely double-stranded. A number of the DNA samples used in this study have been employed as templates for the synthesis of single-stranded RNA polymers the defined nucleotide sequence of which was established by complete nearest-neighbor frequency analyses data). These RNA’s (Nishimura, Jones L%Khorana, 1965; Morgan & Khorana, unpublished directed the incorporation of specific amino acids into polypeptidic material in a cell-free amino acid-incorporating system (Nishimura et al., 1965; Morgan, Wells & Khorana, 1966), providing further evidence for the integrity of the DNA polymers. MAnalytical zone-sedimentation studies were performed in 0.9 M-sodium chloride-O.1 sodium hydroxide solution (Studier, 1965) for each of the polymer preparations used in reported (Wells & Blair, 1967 ; Grant et al., 1968) ; this study. The eosw values previously Morgan & Wells, 1968) are representative of the values observed for these preparations. Thus the double-stranded polymers which contain only two different nucleotides (for example, poly dA * poly dT or poly dIC * poly dIC) have double-stranded molecular weights ranging from 3 to greater than 10 million daltons, the two repeating dinucleotide polymers which contain all four nucleotides have double-stranded molecular weights of 0.4 to 0.8 million daltons and the four repeating trinucleotide polymers have double-stranded molecular weights of 0.2 to 0.4 million daltons. Poly d1 was prepared by separating the strands (Wells & Blair, 1967) of poly d1 epoly dC. Two different preparations of poly dI were used and identical results were found with both preparations. The dITP substrate for the E. coli DNA polymerase-mediated synthesis of the parent poly dI*poly dC was prepared as previously described (Inman & Baldwin, was by preparative paper chromatography in solvent 1964), except that final purification A system (Wells, Jacob, Narang & Khorana, 1967). Spectral and buoyant density analyses in preparation) served to characterize the poly d1; the preparation (Wells et al., manuscript used for most of these studies had an Go,, value of 9.11 s in O-9 M-NaCl-0.1 br-NaOH, indicating a molecular weight of 4-O x lo5 daltons (Studier, 1965). That the poly dI is not a 2-amino group is discussed in the Discussion. contaminated with any purines containing The preparation of poly r1 used in this study has been previously described (Morgan & Wells, 1968). Crab d(A-T) DNA (Smith, 1964) was purified from a crude DNA preparation from Cancer productus (gift of W. Szybalski, University of Wisconsin) by cesium sulfate densityet al., 1965). The ratio gradient centrifugation in the presence of mercury ion (Davidson of HgCl, to total DNA phosphorus was 0.1. Buoyant density experiments as well as spectral analysis on the purified A + T-rich DNA component indicated that no detectable amount (<5%) of the main DNA component (cesium chloride density 1.698 g/cm3) was present.? 1M. Zuteus DNA was isolated by the procedure of Marmur (1961) and the final step was a preparative cesium chloride density-gradient step. The isolated DNA had a buoyant density of 1.723 g/cm3 in an analytical cesium chloride density-gradient and a molecular weight of 3.6 x lo6 daltons (Studier, 1965). Salmon sperm DNA (Worthington Biochemical Corp.) had a molecular weight of 3.2 x lo6 daltons and a cesium chloride buoyant density value of 1.698 g/cm”. (b) Equilibrium

dialysis

Equilibrium dialysis experiments were performed in apparatus designed to contain as little as 0.1 ml. solution on each side of the membrane. The apparatus was constructed as follows: two sheets of Plexiglas (0.5 in. x 2 in. x 7.5 in.) were held together laterally with 7 The purified

A + T-rich

satellite

DNA has a CsCl buoyant density of 1.679 g/cm3 (relative density of 1.425 g/cm3.

E. co& DNA = 1.703 g/cm”) and a CszSOl buoyant

to

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R. D. WELLS

AND

.I. E. LARSON

six thumbscrews. Ten holes (4 mm x 30 mm), centered exactly at the juncture of the two plates, were then drilled; thus, half the hole was in each sheet of Plexiglas. Dialysis tubing (Union Carbide Corp., no. 8) (7.8 cm wide) served as the membrane and was prepared by boiling in 0.01 M-NaCl-0.001 M-EDTA (pH 7.5) for 30 min and then stored at 5°C in deionized water. The dialysis tubing was split down both sides, flattened out and blotted dry. Both face plates of each half of the dialysis apparatus were coated with a light 6lm of stopcock grease at all areas except the holes, the tubing was placed between the plates and the apparatus securely screwed together. To prevent vertical “creep” of the solution, the upper 1 cm of the tubing was greased; the solution never contacted this portion of the membrane. Plastic tape over the top of the cells served to eliminate evaporation; the solution is never in contact with the tape. Control experiments showed that (a) there was no leakage of radioactive solution between the individual cells, and (b) there was no loss of the tritiated actinomycin D due to adsorption to any part of the apparatus or assay system. Plastic micropipets (BioRad Laboratories, Richmond, Cal.) were occasionally used but were found to be unnecessary. DNA solution (0.1 ml.; approximately 1 x 10w5M) was added to one side of each compartment and tritiated actinomycin D solution (0.1 ml. ; 17 x 10m6 to 87 x 10esM) was added to the other side of each compartment (labeled actinomycin D was obtained from Schwarz BioResearch, Inc., spec. act. 3.4 o/m-mole.) At least 20 different experiments, using a 200-fold actinomycin concentration range, were performed with each DNA (Table 1). The solvent for all equilibrium dialysis studies was 0.01 M-NaCl-0.001 M-sodium phosphate (pH 7.4) except where otherwise indicated. The apparatus was shaken gently in the dark at 23 to 24°C; equilibrium was generally achieved in 72 hr as evidenced by repeated sampling of the mixtures at various intervals. However, all experiments were allowed to continue for at least 120 hr before final sampling. Samples (25 ~1.) were taken at various times and added to Bray’s solution (1960) (10 ml.); radioactivity was determined in a Triearb liquid-scintillation counter. The experimental data were treated in the classical manner (Klotz, 1953). Spectral properties of the DNA’s will be reported in a subsequent paper (Wells et al., manuscript in preparation). The extinction of actinomycin D used was 24,800 at 440 nm (Gellert, Smith, Neville & Felsenfeld, 1965). The values listed in Table 1 for K,,, and number of binding sites are reproducible only within k 15%. The assay was designed to detect as little as 1 AM per 100 nucleotides and was successfully used (Table 1) for two DNA’s at this sensitivity. The limit of the sensitivity of this assay is roughly 15 times this value, i.e. 1 AM per 150 nucleotides. (0) Xpectra Ultraviolet and visible spectra were obtained on a Cary 15 recording spectrophotometer with 0 to 0.1 O.D. scale expander. Perturbation spectra were obtained using split-compartment mixing cells (Pyrocell Manufacturing Co.). On mixing just actinomycin and the buffer (no DNA present), a slight hypochromicity was always observed with these cells; also the extent of the hypochromic shift was a function of the presence or absence of divalent metal ions. This behavior is presumably due to the unequal light-path length in the two compartments. All spectra have been corrected for this effect. (d) Analytical

density-gradient

centrifiqation

Analytical cesium sulfate density-gradient centrifugation experiments were performed in a Spinco model E ultracentrifuge as previously described (Morgan & Wells, 1968). All runs were performed at 25’C at 44,770 rev./min. Poly d(A-T) * poly d(A-T) or poly d(I-C) * poly d(I-C) were the density markers used when the binding of AM to another DNA was studied. That neither of these DNA’s binds AM was shown by equilibrium dialysis studies (see Results) as well as by the following two density-gradient experiments. (1) The density of these two DNA’s was determined in the presence of 4 @vr-actinomycin D (Mann Research Laboratories), identical to actinomycin C (Brockmann, 1960), by the isoconcentration distance method of Ifft, Voet & Vinograd (1961) ; the densities found were identical to those determined in the absence of the antibiotic (Wells et al., manuscript in preparation). (2) The position of the DNA band in the centrifuge cell was changed in an identical manner when either buffer or a concentrated actinomycin D solution was added;

BINDING

OF ACTINOMYCIN

D TO DNA

323

that is, two cells were loaded with an identical solution containing only DNA in the cesium sulfate solution and the contents were centrifuged to equilibrium at the same time. Ultraviolet photography established that the DNA bands were in perfect alignment in the two cells. Then 10 ~1. dilute buffer was added to one cell and 10 pl. actinomycin D (to make the final concentration 3.8 pM) was added to the other cell with a Hamilton syringe. Upon re-att&nment of equilibrium, the DNA bands were still in perfect alignment between the two cells, indicating that neither poly d(A-T) . poly d(A-T) nor poly d(I-C) . poly d(I-C) undergoes a buoyant-density shift in the presence of AM. For determining the possible interactions between AM and DNA’s, experiments were done as follows. A cesium sulfate solution (O-65 ml. ; initial density 1.470 g/cm3) containing the DNA (6 to 8 mpmoles) as well as marker DNA was centrifuged to equilibrium. For experiments in which the AM concentration was varied (Figs 8 to lo), after the first oentrito the fugation run, additional concentrated AM solution (5 to 10 PI.)- was added directly centrifuge cell by means of a Hamilton syringe and the DNA buoyant density was again determined. Cesium chloride density-gradient centrifugation was performed 8s previously described (Morgan & Wells, 1968; Wells & Blair, 1967); all density values are relative to E. co&i DNA (density 1.703 g/cm3). (e) RNA

polyrnerase

reactions

The formation of RNA was followed by the incorporation of radioactive ribonucleoside triphosphates into acid-insoluble polynucleotide as previously described (Morgan & Wells, 1968). The reaction mixtures (0.05ml.) contained, per ml.: 40m~-Tris-HCl (pH 8*0), 4 mM-Mgcl,, 1 mM-MnCl,, 12 mM-mercaptoethanol, 500 mpmoles of each ribonucleoside triphosphate (Schwarz BioResearch, Inc.) complementary to both strands of the DNA used as template (unless otherwise designated in the text) (one of the triphosphates bearing a 14C label (Schwarz BioResearch, Inc.)), DNA at the concentration indicated in the legends to the Figures and actinomyoin D (Mann Research Laboratories) at the concentration indicated in the Figures. All components except the enzyme were incubated et 37°C for 15 min; the reaction was then initiated by the addition of the Escherichia coli RNA polymerase. 0.55 mg. RNA polymerase was prepared as previously described (Morgan & Wells, 1968) (2400 units/ml. ; 11 mg/ml.) and was generously provided by Dr A. R. Morgan, Institute for Enzyme Research, University of Wisconsin. Duplicate 20-~1. samples were taken after 30 min and were essayed for acid-insoluble radioactive material (Nishimura, Jacob & Khorana, 1964). Pilot studies with virtually all of the polymeric templates, in which samples were taken at various time intervals up to 60 min, indicated that the 30-min assay point was a valid indicator of the extent of inhibition. A control transcription which contained no AM was performed along with each inhibition reaction to monitor for slight day to day variations in transcription rates; the assays were reproducible within + 10%. (f) General

methods

All glassware, such as cuvettes end pipets, which touched the DNA solutions were soaked in chromic acid and then ammonium hydroxide solution to obviate cross-contamination of the samples. Distilled and then deionized water was used throughout. All pH measurements were made on a Beckman pH meter fitted with a small combination electrode (no. 39030, Beckman Instruments, Fullerton, Calif.). All AM solutions were stored in the dark and care was taken in all experiments to exclude light,

3. Results (a) Equilibrium dialysis A typical binding isotherm demonstrating an actinomycin D-DNA interaction is shown in Figure 1. Experimental data expressed in this manner (Scatchard, 1949) readily provides the number of binding sites per nucleotide from the extrapolated abscissa intercept and the equilibrium constant from the ordinate intercept. The

324

R. D. WELLS

AND

J. E. LARSON

I?IG. 1. Scatchard plot of the binding of actinomycin D to poly d(T-A-C) . poly d(G-T-A). r, Moles bound AM/total moles DNA nucleotide ; A, moles free AM.. The intercept on the abscissa is the number of binding sites per nucleotide and the intercept on the ordinate is the apparent binding constant tinws the number of binding sites. Other details are described in Materials and Methods.

observed equilibrium constants and number of binding sites for ten different DNA’s are listed in Table 1. The equilibrium constant observed for the Micrococcus luteus DNA-actinomycin D interaction is 2.5 x lo6 M-l, and the number of nucleotides per binding site is nine; these results are in excellent agreement with previously reported values (Gellert et al., 1965). Likewise the results observed for salmon sperm DNA (Table 1) are comparable to previous data on similar DNA’s (Gellert et al., 1965; Miiller & Crothers, 1968). The equilibrium constants observed for the interactions with poly d(G-C) . poly d(G-C) and poly dG * poly dC are 3.2 x lo6 and 2.0 x lo6 x-l, respectively; however, almost eight times as much actinomycin is bound to the former DNA TABLE 1 Equilibrium

constants and number of binding sites for DNA-actinomycin interactions

DNA Microoocczcs luteus DNA Salmon sperm DNA Poly d(G-C)*poly d(G-C) Poly dG.poly dC Poly d(T-G)-poly d(C-A) Poly d(T-C)-poly d(G-A) Poly d(T-T-G)-poly d(C-A-A) Poly d(T-T-C)*poly d(G-A-A) Poly $(T-A-C).poly d(G-T-A) Poly dI Binding parameters were obtained under Materials and Methods.

%G+C 72 42 100 100 50 50 33 33 33 from equilibrium

Km, (M-l) 2.5 x 2.0 x 3.2 x 2.0 x 1.3 x 0.6 x 1.2 x 0.7 x 0.8 x 1.3 x dialysis

106 106 10” 106 108 10” 10” 106 106 106

studies;

Nucleotides per site

9 20 12 91 25 37 45 67 50 111 other details

are given

BINDING

OF ACTINOMYCIN

D TO

DNA

325

as to poly dG *poly dC. Our value of 91 nucleotides per binding site for poly dG *poly dC is considerably greater than previously reported values (Kahan, Kahan & Hurwitz, 1963 ; Gellert et al., 1965). The reason for this discrepancy may be that somewhat different experimental techniques were used to derive the data, or it may be due to subtle differences in the preparations of this DNA. The preparations used by both Kahan et al. and Gellert et al. are reported to contain 65% dGMX’ and 35% dCMP; our poly dG . poly dC sample was reconstituted from the previously separated single strands (Inman & Baldwin, 1964; Wells & Blair, 1967). That different authentic preparations of this DNA possess slightly different properties has been recognized (Wells et al., manuscript in preparation; Gray & Tinoco, personal communication). The two repeating dinucleotide DNA’s which contain all four common bases, poly d(T-G) .polyd(C-A)andpolyd(T-C) .polyd(G-A), bindactinomycin(Tablel)somewhat less tightly than naturally occurring DNA’s or the DNA’s containing 100% G + C!; also they bind appreciably less AM. The unpublished data of Hyman & Davidson on poly d(T-G) epoly d(C-A) of K = 2 * 1 x lo6 M-~ and 1 site per 18 & 8 nucleotides are in excellent agreement with our results (Table 1). The same general trend is observed from the data for the two sequence isomeric repeating trinucleotide DNA’s, poly d(T-T-G) *poly d(C-A-A) and poly d(T-T-C) *poly d(G-A-A). Comparing the data for the three pairs of sequence isomeric DNA’s, that is poly d(G-C) *poly d(G-C) versus holy dG *poly dC, poly d(T-G) * poly d(C-A) versus poly d(T-C) * poly d(G-A) and poly d(T-T-G) * poly d(C-A-A) versus poly d(T-T-C) . poly d(G-A-A), the following is observed: (I) the poly d(pur,-pyr,) * poly d(pur,-pyr,) DNA binds more AM than does the poly dpur * poly dpyr sequence isomer; and (2) the former isomer binds AM somewhat more tightly than does the latter DNA, Table 1 shows that poly d(T-A-C) . poly d(G-T-A) binds one AM molecule for each 50 DNA nucleotides with an observed equilibrium constant of 0.8 x lo6 M-I. That the number of binding sites reported here for this DNA is somewhat different from the approximate number previously reported (Wells, 1969) is due to further refinement of the experiments as well as the treatment of the data. The number of sites was previously estimated from a saturation curve (moles AM bound/total moles DNA versus moles free AM) whereas the number of sites are obtained by us from a Scatchard plot. The sequence isomer of this DNA, poly d(A-T-C) *poly d(G-A-T), shows no detectable binding of AM under identical conditions and has been the subject of a previous communication (Wells, 1969). That is, at levels of actinomycin (2 to 3 x 10e6 M) that are saturating for poly d(T-A-C) +poly d(G-T-A) (as well as the other DNA’s in Table 19, approximately 60,000 cts/min/reaction were bound to this DNA; under identical conditions with poly d(A-T-C) * poly d(G-A-T) as the DNA, less than 500 cts/min [3H]actinomycin D per reaction were bound. Five other DNA’s were tested for their ability to bind actinomycin D. None of the following showed detectable binding : poly d(A-T) . poly d(A-T), poly dA . poly dT, poly d(I-C) * poly d(I-C), poly d1 * poly dC and Cancer productus crab d(A-T). Poly d(A-T) . poly d(A-T) and poly d1 * poly dC have been previously reported not to bind AM (Reich & Goldberg, 1964). In addition, poly d(I-C) . poly ld(I-C) induces no change in the visible spectrum of AM under the conditions used by Reich & Goldberg (1964). Hyman $ Davidson (1967) have reported the binding of AM to Cancer antenarrius crab d(A-T) (containing 3% G + C) at a level of one molecule AM per 112 DNA nucleotides. If Cancer productus crab d(A-T) contains as much as 3% G + C, we might have expected detection of a small amount of binding. A possible

326

R.

D. WELLS

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J. E. LARSON

reason for this discrepancy is that different experimental techniques were used as well as somewhat different DNA’s; Hyman & Davidson (1967) used a centrifugation technique for determining binding, whereas we used equilibrium dialysis. A slight amount of binding to Cancer productus d(A-T) is observed using different techniques (see below). (b) Poly dI-actinomycin D complex The most unexpected result of this study is that poly d1 binds actinomyoin D. Equilibrium dialysis studies (Table 1) show that the binding reaction has an apparent equilibrium constant of l-3 x lo6 ~-l and one AM is bound per 111 nucleotides. Indistinguishable results are found when the dialysis solution was made 7 m&rin MgCl, and 1 mna in MnCl, (K = 1-l x lo6 M-~; 99 nucleotides per site). Thus the presence of divalent metal ions is not necessary for complex formation as measured by this assay. In vitro transcription studies also demonstrate that actinomycin D binds to poly d1. l?igure 2 shows that the transcription of poly d1 in the presence of 4.5 j.~M-a

s 5-

3-

s-

-.

I

045 Actinomycin

415

45

26(

D concn (,uM)

FK+. 2. Effect of AM on the transcription of poly d(I-C) . poly d(I-C) and poly d1 by E. coli RNA polymerase. GTP and CTP were present in all reactions; CTP was the 14C-labeled substrate. Template concentrations were: poly d(I-C) . poly d(I-C), 54 pM; poly d1, 35 /UX. In the control experiments (no AM added), the following extents of RNA synthesis were observed in the 30-min time period: poly d(I-C) * poly d(I-C), 2.9-fold; --a--+-, poly d1, 3.0.fold. Other details are -0-O-7 described in Materials and Methods.

proceeds at only 45% of the rate observed in the absence of the antibiotic. At this level of AM, the rate of transcription of poly d(I-C) * poly d(I-C), a DNA that, exhibits no detectable AM binding by equilibrium dialysis, spectral or buoyant-density studies, is virtually identical to that observed in the absence of AM. At higher levels of AM, the transcription of poly d1 is inhibited by as much as 94%. Indeed, this extent of inhibition is at least as great as observed for DNA’s which contain deoxyguanosine (see Pigs 5 to 7).

BINDING

400

OF ACTINOMYCIN

/

450

500

D TO DNA

I

450

327

\. 500

Wavelength (m,d (a)

(b)

FIG. 3. Spectra of aotinomycin D before and after mixing with poly dI. Spectra were obtained with a split-oompartment mixing cell. A composite spectrum was first determined with poly dI(85 F) in one compartment and an equal volume of AM (2.25 pM) in the other compartment (filled circles); the cell was then inverted and the solutions mixed and the perturbation spectra (solid lines) were determined. (a) Solvent was 0.01 M-N&Cl-0.001 M-sodium phosphate-O.007 M-MgC12-O~O01 M-M&l, (all at pH 7.4). (b) Solvent was O-01 M-N&l-0.001 Xsodium phosphate (pH 7.4).

In addition, poly d1 causes a hypochromic shift in the visible spectrum of actinoD. Figure 3 (a) shows that, in the presence of 7 mivr-MgCl,--1 mM-Mm&, 10% hypochromioity is developed at X,,, for actinomycin D (440 mp) . The development of hypochromicity is an immediate reaction; in the time interval necessary to measure the spectrum, the reaction is completed. Figure 3 (b) indicates the necessity of divalent metal ions for rapid complex formation. In the absence of MgCl,-M&l,, only 2% hypochromicity is observed at 440 rnp, and this extent of hypochromicity is unchanged over a 30-minute period. However, if the cuvette is carefully stoppered and kept at 22”C, the slow development of hypochromicity is observed. After four days incubation, the spectrum is indistinguishable from the solid line in Figure 3 (a). In order to observe the binding of aotinomycin D to poly d1 spectrophotometrically, the antibiotic concentration must be relatively low (2.25 or 4.5 PM). If the antibiotic concentration is at a much higher level (22.5 PM), no interaction is observed even in the presence of divalent metal ions; this is due, presumably, to the relatively small amount of AM bound to this DNA (see Table 1). No change in the ultraviolet spectrum of poly d1 is found under any of the above conditions. These results are clearly not in accord with the proposal (Hamilton, Fuller & Reich, 1963; Cerami, Reich, Ward & Goldberg, 1967) that the 2-amino group of deoxyguanosine is necessary for the binding of actinomycin D. The shape of the spectrum of the poly dI-AM complex should not be interpreted as an atypical DNA-AM spectrum. Spectra of identical shape (i.e. X,,,440 to 443 mp) are obtained for the complexes of AM with the following naturally occurring DNA’s: .Micrococcus luteus DNA (G + C = 72%), salmon sperm DNA (G + C = 43%) and Cytophaga johmsonii DNA (G + C = 34%). Thus, for the conditions described in Figure 3, the spectrum of AM is decreased in the presence of a DNA, but is not appreciably shifted to longer wavelengths. If much higher ratios of DNA to AM are used (approximately 40-fold higher than described in the legend to Fig. 3), the typical DNA-AM spectrum (Miiller & Crothers, 1968) is observed. mycin

328

R.D.WELLS

AND J.E.LARSON

It was not possible, for technical reasons, to determine if the presence of AM elevated the T, of poly d1. Assuming that the helical structure of poly d1 is responsible for the binding reaction, the absorbance versus temperature study must be performed in at least 05 M-sodium ion in order for the T, to be greater than 30°C (Inman, 1964). However, Miiller & Crothers (1968) have shown that high salt concentrations lower both the equilibrium constant and the number of binding sites of the DNA-AM complex. Thus when absorbance versm temperature studies were performed on poly d1, in the presence and the absence of AM (22.5 pM) in low salt concentration (O-01Msodium ion), no melting was observed between 15 and 85”C, as expected. When the salt concentration was raised to 1-O M-sodium ion, melting was observed at 47°C as expected (Inman, 1964) ; but the presence of 22.5 PM-AM caused no detectable elevation of the absorbance transition, presumably since the high salt concentration had destablizied the complex. Poly d1 has relatively few binding sites even in dilute salt solution (Table 1). In an effort to overcome the apparent salt destabilization, the AM concentration was raised to 120 ,LLM;however, the large negative solubility coeflicient of AM (Gellert et al., 1965) made this experiment futile. Likewise, when the melting study was performed in low salt concentration in the presence of divalent metal ions (as used in Fig. 3(a)), in the presence and absence of 22.5 PM-~, the polynucleotide formed an insoluble aggregate in both cases at 65°C. Thus it was not possible to determine if the presence of AM elevates the T, of poly d1; in any event, this qualitative measurement is of secondary importance to the equilibrium dialysis studies (see Discussion). To determine if this unexpected binding of 14M to poly d1 is due to a previously unrecognized property of the base inosine, the possible binding of AM by poly r1 was also studied by equilibrium dialysis. Poly r1 binds no detectable AM under identical conditions used for poly d1. (c) In vitro inhibition

of DNA transcription

by actinomycin D

The in vitro inhibition of RNA synthesis by actinomycin is well documented (Goldberg & Rabinowitz, 1962; Hurwitz, Furth, Malamy & Alexander, 1962; Goldberg, Rabinowitz & Reich, 1962; Kahan et al., 1963) ; this effect is the basis for the bacteriostatic and antitumor activities of the molecule (Reich & Goldberg, 1964). Figure 4 shows that the transcription of salmon sperm DNA (42% G + C) is effectively inhibited by AM as has been reported for other naturally occurring deoxyguanine-containing DNA’s; at 4-5 PM-AM, the reaction is inhibited by 89%. Figure 4 also shows the capacity of AM to inhibit the transcription of poly d(A-T) *poly d(A-T) and Cancer productus crab d(A-T). The transcription of neither of these DNA’s is appreciably inhibited except at the high AM levels of 45 and 260 pM. The reaction with crab d(A-T) DNA, which presumably contains approximately 3% G + C (Smith, 1964 and personal communication), is slightly more inhibited than the synthetic polymer which contains no guanine and cytosine. The extent of inhibition observed for this crab d(A-T) DNA agrees well with the data of Widholm & Bonner (1966) on Cancer antenarrius d(A-T) DNA. It was surprising to find that poly d(A-T) *poly d(A-T) transcription was appreciably depressed in the presence of AM. Other studies have demonstrated that this DNA does not bind AM (Goldberg et al., 1962; Kahan et al., 1963; Bhuyan & Smith, 1965; Reich & Goldberg, 1964) and we have confirmed this notion by equilibrium dialysis studies (see above), equilibrium buoyant-density studies and spectral studies. Thus we

BINDING

OF ACTINOMYCIN

0

1

1

I

I

IO.45

329

D TO DNA

45

45

2

Actinomycin D concn (PM)

FIG. 4. Effect of AM on the transcription of salmon sperm DNA, Cancer productzcs crab d(A-T) and poly d(A-T) . poly d(A-T) by E. coli RNA polymerase. All four triphosphates were present when the first two DNA’s were used as templates, and only ATP and UTP were present when poly d(A-T) * poly d(A-T) was the template. 14C-labeled CTP was used to monitor the salmon sperm DNA ternplated reaction and W-labeled ATP was used for the latter two templates. Template concentrations were : salmon sperm DNA, 31 PM; crab d(A-T), 22 pm; poly d(A-T) . poly d(A-T), 45 PM. In the control experiments (no AM added), the following extents of RNA synthesis were observed in the 30-min time period: --A-A---, salmon sperm DNA, 2.3-fold; -o-e-, crab d(A-T), 4.9-fold; -m-m--, poly d(A-T) . poly d(A-T), 4.2-fold. Other details are described in Materials and Methods.

TABLE

Actinomycin D inhibition

DNA

Poly Poly Poly Poly Poly

dI*poly dC dA.poly dT dT dG d(A-T-C).poly

d(G-A-T)

2

of transcription of non-binding DNA’s Percentage of control incorporation in presence of 4.5 PM-AM

Percentage of control incorporation in presence of 260 PM-AM

105 98 108 96 104

43 56 41 74 28

The template concentrations were : poly d1 * pbly dC, 40 pM ; poly dA * poly dT, 33 PM ; poly dT, 30 poly dG, 76 PM; poly d(A-T-C) * poly d(G-A-T), 40 pM. The triphosphates present in the reaction mixtures were : poly d1 * poly dC template, GTP and CTP with GTP ‘W-labeled; poly dA . poly dT template, ATP and UTP with ATP I*C-labeled; poly dT template, 14C-labeled ATP alone; poly dG template, W-labeled CTP alone; poly d(A-T-C) * poly d(G-A-T) template, all four triphosphates with I%-labeled ATP. In control experiments (no AM added), 1*4-, 3*8-, 4*3-, 0.07- and 3*0-fold synthesis, respectively, was observed in the 30-min time-period. Other details are described in Materials and Methods. PM;

330

R. D.

WELLS

AND

J. E. LARSON

examined the capacity of AM to inhibit the transcription of other DNA’s which do not bind the antibiotic. Figure 2 shows that poly d(I-C) epoly d(I-C) transcription is slightly less rapid in the presence of high levels of AM than in its absence. This DNA binds no detectable amount of AM as judged by three criteria (see above). Table 2 shows that the transcription of five DNA’s, which bind no AM physically, is appreciably inhibited at 260 PM-AM. At this AM concentration, approximately a tenfold molar excess of AM per DNA nucleotide is present. At the lower AM level of 45 ,uM, no inhibition is observed; indeed, a slight stimulation is reproducibly observed (also see Fig. 5). Poly dA *poly dT and poly d1 *poly dC do not measurably bind the antibiotic as judged by equilibrium dialysis or buoyant-density studies. Gellert et al. (1965) report that poly dG does not bind AM. The transcription of poly d(A-T-C) * poly d(G-A-T) is discussed below. Thus, the transcription of every DNA we have tested is somewhat depressed in the presence of high levels of AM (see Discussion). Figure 5 shows that AM effectively suppresses the synthesis of poly r(G-C) *poly r(G-C) when poly d(G-C) . poly d(G-C) serves as the template, as expected from the dialysis studies (Table 1). The reaction with the sequence isomer of this DNA, poly dG *poly dC, is considerably less inhibited except at the higher AM levels of 45 and 260 pM. Again, this pattern of inhibition is expected from the data in Table 1; poly dG . poly dC binds less AM and binds less strongly than does poly d(G-C) *poly d(G-C). The influence of AM on synthesis of RNA when poly d(T-G) *poly d(C-A) and its sequence isomer poly d(T-C) . poly d(G-A) serve as templates is shown in Figure 6. Both of these DNA’s contain 50% G + C. Their transcription is markedly inhibited by AM but not to the extent observed for a naturally occurring DNA containing

1

I

0.45 Actinomycin

FIG. 5. Effect of AM on the transcription coti RNA polymerase. Both GTP and CTP were present in all followed. Template concentrations were : d(G-C) . poly d(G-C), 60 pw. In the control respectively, were observed in the 30-min Methods.

I

I

4.5

45

D concn

;

(,uM)

of poly dG * poly dC and poly d(G-C) * poly d(G-C) by E. reactions and the incorporation of 14C-labeled GTP was -.--a---, poly dG.poly dC, 43 PM; -O-O-, poly experiments (no AM added), l.l- and l-9-fold synthesis, time-period. Other details are described in Materials and

BINDING

OF ACTINOMYCIN

D TO DNA

331

Actinomycin D ccwn (,uM)

FIG. 6. Effect of AM on the transcription of poly d(T-G) . poly d(C-A) and poly d(T-C) . poly d(G-A) by E. coli RNA polymerase. All four triphosphates were present in all reactions and the incorporation of 14C-labeled ATP was monitored. Templateconcentrationswere:-~-~-,polyd(T-G)~~polyd(C-A),37~~;-@-~poly d(T-C) . poly d(G-A), 39 PM. In the control experiments (no AM added), 2.3- and 2.6-fold synthesis, respectively, were observed in the 30-min time-period. Other details are described in Materials and Methods.

approximately the same G + C content (see Fig. 4). That poly d(T-G) *poly d(C-A) is more effectively inhibited than poly d(T-C) *poly d(G-A) is consistent with the physical data (Table 1). Marked differences in the extent of inhibition of transcription were observed for the repeating trinucleotide DNA’s (Fig. 7 and Table 2). All four DNA’s have the same base composition (33% G + C) but different nucleotide sequences. The salient features of these data are as follows. (1) The transcription of all four DNA’s is depressed to approximately 20% of control at a high AM level (260 PM) (Fig. 7 and Table 2), but at lower AM levels (4.5 P.BI) striking differences are seen, namely, poly d(T-T-G) . poly d(C-A-A) transcription is most inhibited, then poly d(T-A-C) - poly d(G-T-A), then poly d(T-T-C) spoly d(G-A-A); Table 2 shows that the transcription of poly d(A-T-C) spoly d(G-A-T) is slightly stimulated at 45 ,~M-&bf. (2) The transcription of poly d(T-T-G) . poly d(C-A-A) is appreciably more inhibited than for poly d(T-T-C) *poly d(G-A-A) ; hence, as noted above when comparing sequence isomeric DNA’s, the poly d(pur,-pyr,) *poly d(pur,-pyr,) isomer always binds more AM and binds more tightly than the poly d(pur) *poly d(pyr) isomer. (3) The transcription of poly d(T-A-C) - poly d(G-T-A) IS ’ d’lminished in the presence of AM, as could be predicted from Table 1. (4) The transcription of poly d(A-T-C) *poly d(G-A-T), a DNA which does not bind AM (see above), is unaffected by AM up to 45 p~-.tiM but at higher AM levels is inhibited (Table 2). Similar behavior is observed for other DNA’s which do not bind the antibiotic. However, the inhibition of poly d(A-T-C) - poly d(G-A-T) is slightly greater than seen in the other cases and may represent some non-specific binding of AM at this high concentration (approximately 40 times as much AM as G. C base pairs).

332

R. D. WELLS

0'

I 0.45 Actinomycin

AND

J. E. LARSON

I 45

4h D concn

; >O

(pM)

FIG. 7. Effect of AM on the transcription of poly d(T-T-G) . poly d(C-A-A), poly d(T-A-C) * poly d(G-T-A) and poly d(T-T-C).poly d(G-A-A) by E. COGRNA polymerase. All four triphosphates were present in all reactions and the incorporation of IV-labeled ATP was poly d(T-T-G) . poly d(C-A-A), 37 PM; monitored, Template concentrations were: --w--m--, -.--a---, poly d(T-A-C) . poly d(G-T-A), 52 /AM; -A-A--, poly d(T-T-C) . poly d(G-A-A), 38 PM. In the control experiments (no AM added), 2.8-, 1.8-, and 2*2-fold synthesis, respectively, were observed in the 30-min time-period. Other details are described in Materials and Methods.

In an effort to obtain more information on the molecular site of the binding reaction, we studied the influence of AM on the transcription of individual strands of five different DNA’s. If the AM molecule is bound to both DNA strands, its presence should effect the transcription of both strands. Conversely, if AM is bound to only one DNA strand (for example, the guanine-containing strand), the transcription of one of the DNA strands may be more inhibited than the other. The rate of transcription of each individual strand of five double-stranded DNA’s was studied at four AM concentrations (0.45, 4.5, 45, and 260 PM). The DNA’s and the l*C-labeled ribonucleoside triphosphates (in brackets) studied are : poly dA *poly dT (ATP, UTP) ; poly d1 *poly dC (GTP, CTP); poly dG.poly dC (GTP, CTP); poly d(T-G) .poly d(C-A) (GTP, ATE’); poly d(T-T-G) *poly d(C-A - A) (GTP, ATP). All triphosphates necessary for the synthesis of both strands of each of these DNA’s were always present. In all cases, both strands of each of these DNA’s were inhibited to the same extent (within experimental error of & 10%). (d) Cesium sulfate den&y-gradient

studies

DNA-antibiotic interactions can be qualitatively monitored by equilibrium densitygradient centrifugation studies (Kersten, Kersten $ Szybalski, 1966). However, a major shortcoming of this technique is that, by necessity, the experiments must be performed in concentrated salt solution. Miiller & Crothers (1968) presented dataindicating that the affinity of DNA for AM is decreased by raisingthesalt concentration. Another disadvantage is that the concentrated salt solution may cause unpredictable DNA structural changes, as observed (Wells et al., manuscript in preparation) for poly d(T-C) apoly

BINDING

OF ACTINOMYCIN

D TO

DNA

333

Actinomycin D concn (p M)

FIG. 8. Effect of AM on the cesium sulfate buoyant density of poly d(G-C) . poly d(G-C) and densities were: --a--@-, poly salmon sperm DNA. In the absence of AM, the buoyant salmon sperm DNA, 1.435 g/ems. Other details are d(G-C) . poly d(G-C), 1.448 g/cm3; -A--A---, given in Materials and Methods.

d(G-A) and as suggested below for poly d1. In addition, results from binding studies monitored by this technique may not completely agree with results obtained using other methods. For example, Kersten et al. (1966) showed that denatured DNA undergoes an appreciably greater AM-induced cesium chloride density decrement than does native DNA. We have confirmed this anomalous observation in cesium sulfate gradients; heat-denatured salmon sperm DNA undergoes an AM-induced density shift 15 to 2 times as large as observed for native salmon sperm DNA. However, several laboratories have shown that denatured DNA normally binds somewhat less AM than does the same DNA which was not heated (Reich & Goldberg, 1964). Thus, these experiments must be interpreted cautiously. Figure 8 shows that salmon sperm DNA undergoes a buoyant-density decrement in the presence of AM which is similar to the results reported for &‘arcinu lutea DNA (Kersten et al., 1966). Poly d(G-C) apoly d(G-C) shows a greater densitydecrease than any other DNA studied by this technique including the naturally occurring DNA. These results could be anticipated from the equilibrium dialysis data of Table 1. Because of the anomalous buoyant density behavior of poly dG . poly dC (Wells et al., manuscript in preparation), a meaningful comparison between the two DNA’s which contain lOOo/oG + C is not possible using this assay. The ability of AM to induce a buoyant-density change for other simple DNA polymers was studied. No change in density (within the experimental error of & 1 mg/cm3) was observed in the presence of 3.85 ,uM-actinomycin D for the following DNA’s: poly d(A-T) *poly d(A-T), poly dA . poly dT, poly d(I-C) . poly d(I-C) and poly d1 *poly dC. Cancer productus d(A-T) shows only a small density decrement (4 mg/cm3/mpmole AM/ml.); taken in conjuction with the transcription studies (Fig. 4), this indicates that this DNA binds a small amount of AM although no interaction was observed by

334

R.

D. WELLS

AND

J. E. LARSON

equilibrium dialysis studies. Unexpectedly, poly d1 undergoes no density change in the presence of m (up to 335 PM). Even in the presence of 7 mM-MgCl,-I mM-&Cl,, no density change is observed. As shown above, poly d1 efficiently binds AM as judged by three other criteria. The reason for this behavior is discussed below (Discussion). Figure 9 shows that poly d(T-G) *poly d(C-A) undergoes a buoyant-density shift induced by AM which is virtually identical to that found (Fig. 8) for salmon sperm DNA. The data in Table 1 indicate that these two DNA’s behave similarly. This Figure also shows that the buoyant density of the sequence-isomeric DNA, poly

Actinomycin

D concn (FM)

FIG. 9. Effect of AM on the cesium sulfake buoyant density of poly d(T-G) * poly d(C-A) and poly poly d(T-Qpoly d(G-A). In the absence of AM, the buoyant densities were: --O--O--, poly d(T-C) . poly d(G-A), 1.428 g/cm3. Other d(T-G) * poly d(C-A), 1.422 g/cm 3; -o--o---, details are given in Materials and Methods.

d(T-C) *poly d(G-A), which contains all purines on one strand and all pyrimidines on the complementary strand, is less affected by AM. Again, these experiments buttress the data presented in Table 1; poly d(T-C) +poly d(G-A) binds less AM and binds it less strongly than does poly d(T-G) . poly d(C-A). It should be noted that the magnitude of the density shift observed for poly d(T-C) * poly d(G-A) is not as great as might be expected from Table 1 (compare data for repeating trinucleotide DNA’s and Fig. 10). Figure 10 shows that the repeating trinucleotide DNA’s may also undergo a Cs,SO, buoyant-density shift in the presence of AM. The decrement observed for poly d(T-T-G) * poly d(C-A-A) is not quite so large as seen (Fig. 9) for poly d(T-G) *poly d(C-A); the latter DNA binds appreciably more AM than the former DNA (Table 1). Furthermore, poly d(T-T-G) . poly d(C-A-A) shows a considerably greater actinomycininduced buoyant-density decrement than does poly d(T-T-C) * poly d(G-A-A). Again, this behavior is predictable from the data of Table 1 and is in agreement with the notion that, when comparing sequence isomeric polymers, the poly d(pur,-pyr,) * poly d(pur,-pyr,)DNA binds more AM and binds it more strongly than does the poly d pm . poly d pyr isomer. Poly d(T-A-C) . poly d(G-T-A) binds almost as much AM as does poly d(T-T-G) * poly

BINDING

OF ACTINOMYCIN

335

D TO DNA

Actinomycin D concn (,uM)

FIG. 10. Effect of AM on the cesium sulfate buoyant density of four different repeating trinuoleotide DNA%. poly d(T-T-G) . poly In the absence of AM, the buoyant densities were: -o-o--, d(C-A-A), 1.422g/cm3;-•-•--,poly d(T-A-C) *poly d(G-T-A), 1.422 g/cm”; ---O--O--, poly poly d(A-T-C) . poly d(G-A-T), 1.418 g/cm3. d(T-T-C) * poly d(G-A-A), 1.427 g/cm 3; --n--a--, Other details are described in Materials and Methods.

d(C-A-A) as judged by cesium sulfate buoyant-density experiments (Fig. 10). Equilibrium dialysis studies (Table 1) have shown this to be a valid conclusion. However, the sequence-isomeric repeating trinucleotide DNA, poly d(A-T-C) *poly d(G-A-T), binds virtually no AM as judged by equilibrium dialysis studies (see above). In addition, Figure 10 shows that no change in buoyant density of this DNA is observed in the presence of AM except at quite high concentrations of the antibiotic (4 to 8 moles AM/mole of DNA deoxyguanylic acid). A maximum density decrement of 3 to 5 mg/cm” is observed under these conditions. This observation was the subject of a previous communication (Wells, 1969) and is considered further in the Discussion.

4. Discussion (a) Efect of DNA composition and sequence on binding of actinomycin

D

The three major conclusions that may be drawn from this work are the following : (i) the presence of deoxyguanosine is not necessary for the binding of AM to all kinds of DNA; (ii) the presence of deoxyguanosine is not sufficient for binding; and (iii) a marked nucleotide sequence preference exists for the binding reaction. Each of these conclusions is discussed separately. (i) Non-necessity of quanine for binding Poly d1 binds actinomycin almost as tightly as does naturally occurring DNA as measured by equilibrium dialysis (Table 1) ; also in vitro transcription studies (Fig. 2) in the presence of AM and spectral analysis (Fig. 3) both confirm the binding reaction. Thus, the presence of the 2-amino group on deoxyguanosine, which has been inferred to be necessary for the binding reaction (Hamilton et al., 1963; Cerami et al., 1967), is not essential in all cases. The configuration of poly d1 which is responsible for the binding is unknown; however, from the studies presented above, it seems certain to be an ordered structure as 22

336

R. D. WELLS

AND

J. E. LARSON

opposed to a random coil configuration. Inman (1964) has reported that poly d1 has an ordered structure above room temperature at NaCl concentrations greater than approximately O-2 M. A two-stranded poly d1 structure was suggested by this author. However, it is probable that the ribo analog of this polynucleotide, poly r1, exists as a three-stranded ordered structure (Rich, 19583). Appropriate studies have not yet been performed to determine the exact molecular configuration of the ordered form of poly d1. That an ordered structure of poly d1 is responsible for the antibiotic binding is suggested by the spectral studies (Fig. 3); in the absence of divalent metal ions, no actinomycin spectral change was immediately induced by the polymer, whereas a pronounced spectral change was found in the presence of metal ions. The RNA polymerase inhibition studies (Fig. 2) are also consistent with this suggestion. The transcription of poly d1 was inhibited by AM to a greater extent than any other repeating-sequence polymer tested. These studies must be performed in the presence of divalent metal ions to observe enzymic activity; thus it was not possible to assay for the effect of MgClJVInCl, by this technique. Equilibrium dialysis studies, in either the presence or the absence of divalent metal ions, demonstrated the binding of the antibiotic by poly d1 (Table 1). However, for these studies, AM and the polymer were equilibrated for five to nine days and it is conceivable that the presence of the antibiotic slowly facilitates the formation of a suitable configuration of poly d1 for binding. That this is a slow process, in the absence of MgCl,-M&l,, is suggested by the spectral analyses. Whatever the nature of the structure of poly d1 responsible for the binding reaction, it is clear that a similar structure cannot be formed when the polymer is complexed with poly dC, since poly d1 - poly dC binds no AM as judged by equilibrium dialysis, is vitro transcription or buoyant-densit.y studies. That the sample of poly d1 used for these studies contained some deoxyguanine moieties, which account for the binding, is eliminated by the following considerations. (1) Poly dG shows negligible binding when not complexed with poly dC (Gellert et al., 1965). (2) The method of preparation of poly d1 would exclude the presence of deoxyguauine residues (see Materals and Methods). dATP was deaminated with nitrous acid to provide the substrate, dITP, which was purified chromatographically from any unreacted dATP. Even if the starting dATP was contaminated with trace amounts of dGTP, which were undetectable (less than 2%) in the chromatography systems used, the product would be deoxyxanthosine triphosphate. This analog is very poorly incorporated by the DNA polymerase (Bessman et al., 1958). (3) Other DNA polymers (poly d1 * poly dC and poly d(I-C) - poly d(I-C)) used in this study were synthesized with the same dITP sample as that used for the formation of poly d1. If the dITP preparation contained a small amount of dGTP, some percentage of the G-containing analogs of these DNA’s would have been formed (poly dG - poly dC and poly d(G-C) . poly d(G-C), respectively). That neither of the former DNA’s binds AM, whereas both of the latter DNA’s do bind, indicates that the dITP sample was uncontaminated. Complex formation between AM and poly dI was not detectable by equilibrium buoyant-density centrifugation studies. A possible reason for this anomalous behavior is the lack of sensitivity of this assay. High salt concentrations are known (Miiller & Crothers, 1968) to lower the equilibrium constant for the DNA-AM complex and, in addition, decrease the number of effective binding sites. Poly d1 binds less AM than any of the DNA’s tested (Table 1). Poly d(T-T-C) -poly d(G-A-A) has 1.7 times as many binding sites as does poly d1; the maximum density decrement observed for

BINDING

OF ACTINOMYCIN

D TOIDNA

337

this DNA was only nine times the experimental error ( & 1 mg/cm3). In contrast, poly d(T-A-C) *poly d(G-T-A) has about the same number of sites as poly d(T-T-C) *poly d(G-A-A) (1.3 times as many), but this DNA shows a marked AM-induced density decrement. This indicates that the concentrated salt solution may affect various DNA’s somewhat differently. That these two repeating trinucleotide DNA’s probably have different configurations will be the subject of a subsequent communication (Wells et al., manuscript in preparation). Previous work (Inman, 1964) suggests that the ordered structure of poly dI should be stabilized by high salt concentration. However, some multistranded polymer complexes are known to be destroyed at very high salt concentrations (Rich, 1958a; Davies & Rich, 1958). Thus, since concentrated salt solutions may have somewhat different effects with the various DNA’s, and since poly d1 shows a relatively small number of binding sites (even in 0.01 M-Na+), this apparently anomalous effect can be understood. Other pitfalls in interpreting AM-DNA complex formation studies by density-gradient centrifugation studies were alluded to in Results. (ii) Insuficiency

of guanine for binding Poly d(A-T-C) - poly d(G-A-T), which contains 33% G + C, binds virtually no AM as measured by equilibrium dialysis, spectral, absorbance-temperature, in vitro transcription and buoyant-density studies (Wells, 1969 and above). Thus, contrary to predictions from previous studies (Cerami et al., 1967; Reich & Goldberg, 1964), the presence of guanine in a DNA is not a su%icient requisite for binding. The role of G - C pairs in influencing the binding with other DNA’s is in no way contradicted by this finding. In fact the bulk of the studies in this paper (Table 1) buttress the generally accepted observation that G * C pairs have some role in the binding reaction. The reason that poly d(A-T-C) - poly d(G-A-T) does not bind is unknown at present. However, physical and enzymic studies (Wells, Jacob, Narang & Khorana, 1967 ; Wells & Blair, 1967; Wells et al., manuscript in preparation) show that this DNA has somewhat dilferent properties from its sequence isomer, poly d(T-A-C) * poly d(G-T-A), which does bind AM (Table 1, Figs 7 and 10). Hence, poly d(A-T-C) *poly d(G-A-T) must possess a molecular configuration which does not permit AM binding. What, then, is the role of deoxyguanine in the binding reaction ? It was suggested (Wells, 1969) that the presence of deoxyguanosine in a DNA may induce a suitable configuration in a small region of the DNA chain to permit binding. Thus, it was possibly fortuitous that, in all previous studies (Cerami et al., 1967 ; Reich & Goldberg, 1964), polymers which contain a purine 2-amino group do bind AM whereas polymers without a purine 2-amino group do not bind AM. That is, for all DNA’s examined to date except one (poly d(A-T-C) epoly d(G-A-T)), the presence of a purine 2-amino group was sufficient to generate an appropriate configuration for the binding of AM. We are suggesting that, in most cases, the presence of G in a DNA is sufficient to generate an appropriate configuration; however, the presence of G is not necessarily sufficient or obligatory. For example, in certain cases (namely, poly d1) a suitable configuration may be formed without the presence of G. The details of the molecular nature of the appropriate configuration for binding are obscure at present. Indeed, such information may only be obtained by X-ray diffraction analyses of a variety of DNA’s or of a suitable AM-polynucleotide complex.

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(iii) Nucleotide sequencepreference for binding The majority of the studies reported were performed with DNA polymers with defined repeating nucleotide sequences. By complete nearest-neighbor frequency analyses it was possible to assign with certainty the primary nucleotide sequence in each case (Radding et al., 1962; Byrd et al., 1965; Wells et al., 1965; Wds, Jacob, Narang & Khorana, 1967; Grant et al., 1968). Thus it was possible to study the influence of DNA nucleotide sequence on the binding reaction. Equilibrium dialysis studies (Table l), as well as all other studies reported here, indicate that in comparing sequence-isomeric DNA’s, the isomer which contains both purines and pyrimidines on both complementary strands binds more AM and binds it more tightly than does the isomer which contains all purines on one strand and all pyrimidines on the complementary strand. Thus poly d(G-C) . poly d(G-C), poly d(T-G). poly d(C-A) and poly d(T-T-G) * poly d(C-A-A) bind more AM and bind it more tightly than do poly dG * poly dC, poly d(T-C) . poly d(G-A) and poly d(T-T-C) *poly d(G-A-A) respectively. Such a comparision is not possible with poly dA. poly dT, poly d(A-T) *poly d(A-T), poly d1 * poly dC and poly d(I-C) =poly d(I-C), since none of these polymers binds AM. A variety of physical and enzymic data indicates that the poly d pur . poly d pyr isomer is somewhat anomalous when compared to the poly d(pur,-pyr,) . poly d(pur,pyr,) isomer and to naturally occurring DNA. Thus, whether the apparent DNA specificity demonstrated here is a function of primary nucleotide sequence or a matter of DNA configuration cannot be ascertained at present. Indeed, it may be that these two variables are inseparable; hence such a discussion would be meaningless, since it appears that the DNA nucleotide sequence determines the three-dimensional configuration (Wells et al., manuscript in preparation; Mitsui, Langridge & Wells, 1969). Unquestionably the most marked effect of nucleotide sequence was observed in comparing poly d(T-A-C) *poly d(G-T-A), which binds AM, with poly d(A-T-C) *poly d(G-A-T), which does not bind AM. A longer abbreviation for these DNA’s is: poly d(T-A-C) .poly d(G-T-A) - - 5’-end 3’-end - - -ATGATGATGAT5’-end ---TACTACTACTA--3’-end poly d(A-T-C) * poly d(G-A-T) 3’-end - - -TAGTAGTAGTA- - 5’-end 5’-end - - -ATCATC ATCAT- - - 3’-end Thus, focusing on a G. C pair in both DNA’s, the difference is simply the relative orientation of the A * T pairs. The binding properties of these two DNA’s are discussed above. Previous studies on the binding of AM to DNA have indicated the importance of nucleotide sequence. Gellert et al. (1965) found that the stoichiometry of the binding reaction was relatively constant for a variety of naturally occurring DNA’s, ranging in composition from 35 to 73% G + C. Also, the studies of Hyman & Davidson (1967) on the binding of AM to the Cancer antenarrius crab d(A-T) DNA suggest a sequence specificity. Indeed, such a specificity has been established for the binding of nogalamycin (a tetracycline-like molecule) to DNA (Bhuyan & Smith, 1965). (b) Techniques for studying the binding reaction The five different techniques used for studying the binding of AM to DNA in this and the previous (Wells, 1969) communication are equilibrium dialysis, in vitro inhibition of transcription, spectral analysis, elevation of melting temperature and

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buoyant-density centrifugation studies. We consider the equilibrium dialysis experiments (Table 1) to be the most reliable and thermodynamically rigorous studies reported. Results obtained using the other four techniques are important only because they complement, and are usually consistent with, the equilibrium dialysis studies. Visible spectral changes induced in AM by a binding DNA suffer from the drawback that the spectral characteristics of each of the multiple modes of binding has not been elucidated; hence they must, at present, be assumed to be identical. This assumption is probably unfounded. Analytical buoyant-density gradient centrifugation with DNA-AM complexes in concentrated salt solutions clearly is not a rigorous and reliable experimental technique (see above). Indeed, in at least two oases (denatured DNA verse native DNA binding and poly d1 binding), results obtained by this method do not agree with results obtained by other methods. The interpretation of transcription studies with the Escherichia COGRNA polymerase in the presence of AM is complicated by the following considerations. (1) The system is multicomponent and any of a variety of steps may be inhibited by the antibiotic (Richardson, 1966aJ; Maitra, Cohen & Hurwitz, 1966; Sentenac, Simon & Fromageot, 1968). (2) The transcription of 17 different DNA’s was studied, including seven DNA’s which do not bind AM as judged by other techniques. The transcription of all of these DNA’s was inhibited if the AM concentration was sufficiently great. Hence, these studies are only useful when they are analyzed together with a wide variety of other studies performed in parallel. The transcription studies reported reveal some previously unrecognized features. (1) The transcription of all seventeen DNA’s studied is inhibited at sufficiently high AM concentrations. That the transcription of non-binding DNA’s was inhibited may be due (a) to a non-specific inhibition of the enzyme, or (b) to the enzyme facilitating the binding of AM to a DNA which does not normally bind the antibiotic under other conditions. The former explanation is deemed unlikely since the extent of inhibition of the various non-binding DNA’s is not identical (Table 2, Figs 2 and 4). This effect clearly warrants further study and may be related to the different modes of binding of the antibiotic (Gellert et al., 1965; Mtiller & Crothers, 1968; Cavalieri & Nemchin, 1968). (2) The extent of inhibition of transcription is apparently not only a function of the amount of AM bound to a DNA but also of the complexity of the nucleotide sequence of the DNA. The transcription of salmon sperm DNA (G + C = 43%) is considerably more inhibited than the transcription of poly d(T-G) +poly d(C-A) (G + C = 50%) (Figs 4 and 6). Likewise, the transcription of poly d(T-T-G) . poly d(C-A-A), and to a lesser extent poly d(T-A-C) - poly d(G-T-A) (G + C = 33% for both), is more inhibited than the transcription of poly d(T-G) epoly d(C-A). Thus, although the repeating dinucleotide DNA binds more AM than the repeating trinucleotide DNA’s, the latter DNA’s with the more complex sequence, are somewhat more inhibited. Other studies (Wells & Blair, 1967; Wells et al., manuscript in preparation) indicate that the properties of the repeating trinucleotide DNA’s are more closely related to those of naturally occurring DNA’s than for the repeating dinuoleotide DNA%. (3) The extent of inhibition of transcription observed for the repeating polymer DNA’s is appreciably less than found for naturally occurring DNA’s with similar nucleotide compositions. This may be due to the polymer-AM complex dissociating more rapidly than the naturally occurring DNA-AM complex. Indeed, Muller & Crothers (1968) have found that the poly dG *poly dC complex dissociates approximately ten times faster than the latter complex.

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The techniques employed are discussed, since a variety of methods were used with a greater number of DNA’s than studied before and since at least part of the problem of establishing the nature of the AM-DNA complex (Waring, 1968a) is apparently a matter of the experimental techniques and conditions used by different laboratories. (c) Molecular nature of DNA-actinomycin

D complex

The molecular nature of the complex formed between AM and DNA has been the subject of considerable study (Reich & Goldberg, 1964; Gellert et al., 1965; Cerami et al., 1967; Mtiller & Crothers, 1968). Two models have been proposed. (1) Hamilton et al. (1963) proposed that the AM chromophore is hydrogen-bonded to the outside of the DNA helix; stabilization of the complex is provided by a hydrogen bond between the actinomycin quinone oxygen and the S-amino group of guanine as well as hydrogen bonds from the AM amino group to the N(3) of guanine and to the deoxyribose ring oxygen. The peptide la&ones were considered to provide additional hydrogen-bonds with phosphodiester oxygens. The major justifications for the proposal were: (a) the apparent specificity for deoxyguanosine residues, (b) the importance of an rmsubstituted chromophore amino group, (c) the inability to detect intercalation by X-ray diffraction studies (Reich et al., 1967). (2) Muher & Crothers (1968) proposed that the AM chromophore is intercalated into the DNA chain with the peptide lactones projecting into the DNA minor groove. The basis for this proposal was : (i) the marked decrease in the rate of complex formation when AM analogs contained a bulky substituent on the 7 position of the chromophore (the side of the chromophore distal to the helix in the above model) and (ii) a careful re-examination of the hydrodynamic properties of DNA of various molecular weights in the presence of AM. Recent studies by Waring (19688) are consistent with the intercalation model ; actinomyoin acts virtually identically to ethidium (an acknowledged intercalating agent) in its effect on the supercoiled structure of the replicative form of +X174 DNA. A complete review of the justification for each of these models cannot be presented here and the reader is referred to the references cited. That multiple modes of binding exist has been established by virtually all laboratories concerned with this problem (Reich & Goldberg, 1964; Gellert et al., 1965; Miiller & Crothers, 1968; Cavalieri & Nemchin, 1968). Hence, when discussing the molecular nature of the complex, it should be clear that only the very tight binding of AM to DNA is considered. As mentioned above, one reason why Hamilton et al., (1963) proposed the hydrogenbonded “outside-binding” model was the apparent necessity of deoxyguanine for binding. Apurinic acid, poly d(A-T) - poly d(A-T), poly dA - poly dT and poly d1 *poly dC do not form complexes with AM (Reich et al., 1967). Conversely, DNA’s which contain deoxyguanine, or a purine bearing a 2-amino group, do bind AM and include naturally occurring DNA, poly dG +poly dC, poly d(DAP-T) * poly d(%@-T) (Cerami et al., 1967), a d(A-T)-like polymer containing a proportion of 2-aminopurine residues (Reich et al., 1967) and Cancer antenarrius crab d(A-T) (Hyman & Davidson, 1967). Hence it was suggested that the presence of a purine a-amino group is sufbcient, and perhaps the sole requirement, for AM binding (Cerami et al., 1967). Our results are clearly not in agreement with this suggestion, since poly d1 binds AM (Table 1) and inosine bears no e-amino group. It is, of course, possible that the mode of binding of AM by poly d1 bears little relationship to the mode of binding by a naturally occurring DNA containing a two-stranded Watson-Crick structure. However, at least two of the polymers in Table 1 (poly dG - poly dC and poly d(T-C) - poly d(G-A) ) do not

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have common DNA configurations but do bind the antibiotic. Furthermore, our results do not indicate that the presence of deoxyguanine (or a purine 2-amino group) is a sufficient requirement for binding, since poly d(A-T-C) *poly d(G-A-T), which contains 33% G + C, binds no AM (see above and Wells, 1969). We propose that the apparent specificity of guanine (or a purine 2-amino group) previously observed, and corroborated in some oases in this paper, is a secondary effect. That is, we propose that the presence of a purine S-amino group in a polymer is sufficient, in most cases, to confer on a DNA a suitable steric and electronic environment to permit the binding of AM. Thus, the presence of a suitable DNA configuration may be the determinant for AM binding rather than the presence of a purine 2-amino group. That poly d(A-T-C) - poly d(G-A-T) does not bind AM is because, although it contains G *C pairs, other structural forces do not allow it to assume a suitable configuration for binding (Wells et al., manuscript in preparation). We suggest that other DNA’s will be found that contain G but do not bind AM, and also DNA’s that do not contain G but do bind AM. The molecular nature of the hypothetical binding environment is unknown at present. One feasible structure, which is consistent with our results, has been proposed (Mi.iller & Crothers, 1968) on the basis of model building. This work has been supported by grants from the National Science Foundation (GB6629), the Life Insurance Medical Research Foundation and the Wisconsin Alumni Research Foundation. We thank Drs M. Gellert and G. Felsenfeld for sending us their equations for determining binding parameters from spectrophotometric titration data, and Dr J. Anderegg for the use of his microdensitometer. REFERENCES Arnott, S., Wilkins, M. H. F., Fuller, W. & Langridge, R. (1967). J. Mol. Biol. 27, 535. Bessman, M. J., Lehman, I. R., Adler, J., Zimmerman, S. B., Simms, E. S. & Kornberg, A. (1958). Proc. Nat. Acad. Sci., Wash. 44, 633. Bhuyan, B. K. & Smith, C. G. (1965). Proc. Nat. AC&. Sci., Wash. 54, 566. Bray, G. A. (1960). Analyt. Biochem. 1, 279. Brockmann, H. (1960). In Fortschritte der Chemie organ&her NaturstofSe, vol. 18, p. 1. Vienna : Springer Verlag. Byrd, C., Ohtsuka, E., Moon, M. W. & Khorana, H. 0. (1965). Proc. Nat. Acud. Sci., Wash. 53, 79. Cavalieri, L. F. & Nemchin, R. G. (1968). Biochim. biophys. Acta, 166, 722. Cerami, A., Reich, E., Ward, D. C. & Goldberg, I. H. (1967). Proc. Nat. Acad. Sci., Woxh. 57, 1036. Davidson, N., WidhoIm, J., Nandi, U. S., Jensen, R., Olivera, B. M. & Wang, J. C. (1965). Proc. Nat. Acad. Sci., Wash. 53, 111. Davies, D. R. & Rich, A. (1958). J. Amer. Chem. Sot. 80, 1003. Gellert, M., Smith, C. E., Neville, D. & Felsenfeld, G. (1965). J. Mol. Biol. 11, 445. Goldberg, I. H. & Rabinowitz, M. (1962). Science, 136, 315. Goldberg, I. H., Rabinowitz, M. & Reich, E. (1962). Proc. Nat. Ad. Sk., Woxd. 48,2094. Grant, R. C., Harwood, S. J. & Wells, R. D. (1968). J. Amer. Chem. Sot. 90, 4474. Hamilton, L. D., Fuller, W. & Reich, E. (1963). Nature, 198, 538. Haselkorn, R. (1964). Science, 143, 682. Hurwitz, J., Furth, J. J., Malamy, M. & Alexander, M. (1962). Proc. Nat. Acad. Sci., Wash. 48, 1222. Hyman, R. W. 8: Davidson, N. (1967). Biochem. Biophys. Res. Comm. 26, 116. II%, J. B., Voet, D. H. & Vinograd, J. (1961). J. Phys. Chem. 65, 1138. Inman, R. B. (1964). J. MOE. Biol. 9, 624. Inman, R. B. & Baldwin, R. L. (1964). J. Mol. BioZ. 8, 452. Kahan, E., Kahan, F. & Hurwitz, J. (1963). J. BioZ. Chem. 238, 2491.

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