The interaction of daunorubicin and doxorubicin with DNA and chromatin

206

Biochimica et Biophysica Acta, 607 (1980) 206--214 © Elsevier/North-Holland Biomedical Press

BBA 99645

THE INTERACTION OF DAUNORUBICIN AND DOXORUBICIN WITH DNA AND CHROMATIN

FRANCO ZUNINO, AURELIO DI MARCO, ADRIANO ZACCARA and ROMOLO A. GAMBETTA

Division o f Experimental Oncology B, Istituto Nazionale per lo Studio e la Cura dei Tumori, Via Venezian 1, 20133 Milan (Italy) (Received July 13th, 1979)

Key words: Daunorubicin; Doxorubicin; DNA-drug binding; DNA intercalation; Chromatindrug binding; Anthracycline antibiotic

Summary Isotherms that describe the binding of anthracycline antibiotics (including daunorubicin and doxorubicin (adriamycin)) to calf thymus DNA and chromatin have been obtained by means of fluorescence measurements. As expected for charged ligands, the association constants for the interaction of all drugs examined with DNA were found to be dependent on the ionic strength. However, in the case of the daunorubicin-DNA interaction, a marked decrease in the number of binding sites was also observed when the ionic strength was increased. It is suggested that the effect of salt concentration on the number of potential binding sites of daunorubicin molecules to DNA may be the result of some salt-induced alterations in the DNA conformation. This interpretation is also supported by binding data obtained with calf thymus chromatin. Whereas at low salt concentration the binding parameters for the doxorubicin-chromatin interaction are similar to those expected by neutralization of the phosphate groups by histones, modifications of the DNA structure in chromatin are invoked to account for the reduction and heterogeneity of daunorubicin binding sites. The side chain at C-9 could play an important role in determining the strength and specificity of the anthracycline-DNA interaction. Introduction A great variety of substances, including several agents of importance in cancer chemotherapy [1], are known to bind to DNA by intercalation [2]. Abbreviation: SDS0sodium dodecyl sulphate.

207 Attention has been concentrated on the classical intercalating drugs, acridines and ethidium bromide [2--4]. Studies on the binding of various dyes, drugs and antibiotics to DNA and chromatin have contributed to an understanding of the structure of these macromolecules [4--12] and have suggested possible mechanisms of the biological activity of some drugs [1]. Molecular models of the intercalation of some drugs into DNA have been described [2], and it is already apparent that differences in the binding specificity must be expected [13]. Indeed, different intercalating agents produce different effects on DNA [4]. It remains to be elucidated if such differences may influence their selectivity in drug action. The interaction of daunorubicin * and doxorubicin * (the main representatives of the anthracycline group of antibiotics) with DNA is of general interest in connection with questions regarding drug-nucleic acid interactions, since their biological activity is thought to reside in the ability to bind to DNA [14], and highly stereospecific requirements in both DNA binding ability and biological activity of the natural drugs has been reported [15--18]. The increased therapeutic effectiveness of doxorubicin compared to daunorubicin [14] has also created an interest in comparing the DNA binding properties of these drugs [15,19,20]. Non-intercalated substituents on the chromophore or away from the chromophore are believed to significantly contribute to the binding process of intercalating agents [5,16,21], but relatively little concrete information is available as to how they are involved, at least in the case of anthracycline antibiotics [ 19]. The aforementioned studies serve to emphasize the need for more information about the factors which control the binding of anthracyclines to DNA. The present work is a further evaluation of the DNA binding properties of daunorubicin and doxorubicin under different experimental conditions. In addition, the drug binding studies were extended to isolated chromatin to determine the influence of chromosomal proteins on the antibiotic-DNA interaction. Anthracycline binding to chromatin has been reported by cytological studies using fluorescence or autoradiography [14,22]. Materials and Methods Antibiotics. Daunorubicin, doxorubicin and derivatives were supplied by Farmitalia (Milan, Italy). The antibiotics were stored in the dark in a desiccator at 4°C. Drug solutions were freshly prepared immediately before use. Their homogeneity had been checked in the Farmitalia laboratories; the drugs were used without further purification. Isolation o f chromatin. Nuclei from calf thymus were prepared essentially according to the procedure of Allfrey et al. [23], except that 3 mM MgC12 was substituted for 3 mM CaC12. The nuclei were resuspended in 0.25 M sucrose and 3 mM MgC12 at a final concentration of about 108 nuclei/ml. For isolationof chromatin nuclei were washed [24] once with 0.6 M sucrose (containing 0.05 M Tris-HC1, pH 7.0, 0.025 M KC1 and 5 mM MgCl~) and three times with

* Daunorubicin

and doxorubicin

are synonymous

with daunomycin

and adriamycin, respectively.

208 0.01 M Tris-HC1, pH 8.0, 0.15 M NaC1 and 0.2 mM EDTA. Nuclei were gently lysed in 0.2 mM EDTA, pH 7.0, in an ice bath; shearing was avoided by repeated suspension and centrifugation in 0.2 mM EDTA. The gel-like pellet obtained after centrifugation in 0.2 mM EDTA was gently resuspended in 0.05 mM EDTA and stored at 0°C for 24--48 h. The partially solubilized gel was centrifuged at 5000 rev./min for 15 min. The supernatant was dialyzed at 0°C against 0.01 M Tris-HC1 (pH 7.0), 0.01 M NaC1 and 0.5 mM EDTA for binding experiments. Chromatin prepared in the presence of 0.1 mM phenylmethylsulphonyl fluoride gave similar results. All samples used had a protein to DNA ratio of 1.50 -+ 0.10 g/g. Preparation of DNA. Calf thymus DNA was prepared according to Thomas et al. [25]. Purified nuclei were lysed in 0.5% SDS and deproteinized by pronase treatment and phenol extraction. The phenol was removed by dialysis against 0.01 M Tris-HC1 (pH 7.0) and 0.5 mM EDTA. This DNA sample had an A 2 6 o n m / A 2 8 o n m ratio of 1.87, a n A 2 6 0 n m / A 2 3 0 n m ratio of 2.4, and a total hyperchromicity of 260 nm of 37% (at an ionic strength of 0.01). The same preparation of native calf thymus DNA was used in all experiments. PM2 DNA was prepared according to the method of Espejo et al. [26] from purified bacteriophage [27]. DNA concentrations (expressed in terms of molar concentration with respect to nucleotides) were determined using a molar extinction coefficient of 6600 M -1 • cm -1 at 260 nm. Binding measurements. Fluroescence studies of the binding of anthracycline antibiotics to DNA and chromatin were carried out using a Perkin-Elmer model MPF 44A spectrofluorometer. The method for determining the binding parameters from fluorescence quenching [3] has been described [15]. The excitation wavelength was 470 nm, and emission was monitored at 592 nm. All measurements were carried out at 20°C in 0.01 M Tris-HC1 (pH 7.0) and 0.5 mM EDTA plus concentrations of NaC1 as indicated. At the chromatin concentrations used, no effect of light scattering was usually detected. The binding data were analyzed by the Scatchard method [28]. Results and Discussion A characteristic property of the association constants for positively charged molecule-nucleic acid interactions is the substantial dependence on ion concentrations [29--31]. The electrostatic component of the binding reaction is of crucial importance in the case of the anthracycline-DNA interaction due to the highly stereospecific requirements of the 3'-amino group at the DNA binding site [16,18]. As expected, the association constant for the doxorubicin-DNA interaction is strongly dependent on ionic strength [16]. However, a reverse effect has been reported for the daunorubicin-DNA interaction [ 32]. Although this discrepancy may, at least in part, reflect differences in experimental conditions, it is of interest to reexamine the effect of the ionic strength on the binding of these antrhacyclines to DNA under identical experimental conditions. The binding parameters (Table I) obtained in the linear region of the Scatchard plot at low r values (Fig. 1) show a decrease in the binding constant as a function of the ionic strength for both antibiotics. However, at each ionic

209

TABLE I BINDING PARAMETERS FOR THE INTERACTION OF DAUNORUBICIN W I T H C A L F T H Y M U S D N A A T V A R I O U S NaCI C O N C E N T R A T I O N S

AND DERIVATIVES

K a p p a n d n w e r e d e t e r m i n e d f r o m S c a t c h a r d p l o t s (see l e g e n d t o Fig. 1). All e x p e r i m e n t s w e r e c a r r i e d o u t at 2()°C in 0 . 0 1 M Tris-HC] ( p H 7 . 0 ) , 0 . 5 m M E D T A a n d v a r i o u s c o n c e n t r a t i o n s o f NaCI as i n d i c a t e d . All d a t a w e r e d e r i v e d f r o m f l u o r e s c e n c e q u e n c h i n g studies. See Materials a n d M e t h o d s f o r details. E a c h v a l u e r e p r e s e n t s a n a v e r a g e o f a t least t h r e e i n d e p e n d e n t d e t e r m i n a t i o n s . NaCI concentration (M)

0.01 0.10 1.0

Daunorubicin

Doxorubicin

K a p p ( M -I )

n

1 2 . 2 " 106 4 . 8 " 106 1,31 - 106

K a p p ( M -I )

0.220 0.160 0.081

19.1 " 106 6.5 " 106 1 . 5 8 " 106

Daunosaminyldaunorubicin

n

0.212 0.179 0.150

K a p p ( M -l )

n

9.8 " 106 2.7 " 106 0 . 3 8 - 106

0.358 0.283 0.250

strength the association constant for doxombicin is higher than that observed for daunorubicin. The number of binding sites also decreased from 0.22 at 0.01 M NaCl to 0.081 at 1 M NaCI for the daunorubicin-DNA binding. In contrast, in the case of the doxorubicin-DNA interaction, the number of binding sites changed littleas previously reported [16]. Although the binding constants determined in this study were significantly higher than those previously obtained (presumably due to differences in the D N A preparation), the order determined for the D N A binding constants of daunorubicin and doxorubicin was in good agreement with the value obtained using the same method [15,16,

19]. A

\

B

\

L t '

dl

d2

o

'

d~

o12

Fig. 1. S c a t c h a r d p l o t s f o r d o x o r u b i c i n b i n d i n g t o calf t h y m u s D N A ( A ) a n d PM2 c l o s e d c i r c u l a r D N A (B). E x p e r i m e n t s w e r e c a r r i e d o u t a t 2 0 ° C in 0 . 0 1 M Tris-HCl ( p H 7.0), 0.1 M NaCI a n d 0 . 5 m M E D T A . All d a t a w e r e d e r i v e d f r o m f l u o r e s c e n c e q u e n c h i n g studies. See Materials a n d M e t h o d s f o r details. T h e e x p r e s s i o n r/m = K a p p ( n - - r ) is u s e d t o c o n s t r u c t a S c a t c h a r d p l o t , w h e r e r is t h e m o l a r r a t i o o f b o u n d a n t i b i o t i c p e r n u c l e o t i d e a n d m is t h e m o l a r c o n c e n t r a t i o n o f free a n t i b i o t i c . In this Plot, K a p p ( t h e a p p a r e n t b i n d i n g c o n s t a n t ) is t h e n e g a t i v e o f t h e s l o p e a n d n (the a p p a r e n t n u m b e r o f b I n d i n g sites p e r n u c l e o t i d e ) is t h e i n t e r c e p t o f t h e l i n e a r r e g i o n o f t h e b i n d i n g c u r v e w i t h t h e h o r i z o n t a l axis. T h e v a l u e s o f K a p p a n d n r e f e r In all cases ( T a b l e s l - - I I l ) t o a n e a r l y l i n e a r r e g i o n o f t h e b i n d i n g i s o t h e r m a t s m a l l v a l u e s o f r. F o r t h e d a u n o r u b i c I n - P M 2 D N A i n t e r a c t i o n , a n e a r l y l i n e a r r e g i o n o f t h e b i n d i n g i s o t h e r m is u s u a l l y f o u n d a t l o w e r v a l u e s of r (less t h a n 0 . 1 ) t h a n f o r d o x o r u b i c I n .

210

i -2

-4

6

log I Fig. 2. D e p e n d e n c e o f t h e a p p a r e n t b i n d i n g c o n s t a n t ( K a p p ) f o r t h e i n t e r a c t i o n o f a n t h r a c y c l i n e s w i t h D N A o n t h e i o n i c s t r e n g t h (I) in a d o u b l e - l o g a r i t h m i c p l o t . o, d o x o r u b i c i n ; e, d a u n o r u b i c i n ; ×, d a u n o s a m i n y l - d a u n o r u b i c i n . See T a b l e I f o r d e t a i l s .

If the logarithm of the binding constant is plotted versus the logarithm of the ionic strength (/) (Fig. 2), a straight line is obtained for both antibiotics, with slopes of 0.58 and 0.65 for daunorubicin and doxorubicin, respectively. The linear relationship between log gap p and log I is predicted on the basis of the theory of ion effects on ligand-nucleic interactions [29,30]. However, the slopes are slightly lower than those reported for other charged ligands [ 30,33, 34]. The influence of a monovalent cation concentration in the DNA binding of a cationic ligand is expected, since competition with salts as counterions for the phosphate groups plays a dominant part in dictating the thermodynamic aspects of the interactions [30,31]. The higher slope found for daunosaminyldaunorubicin (which contains two charges) is also understandable in terms of a competition model. However, since both daunorubicin and doxorubicin contain only one charge, the strikingly different effect of salt concentration on the number of binding sites of the two antibiotics is n o t simply explained by polyelectrolyte behavior. Since the conformational properties of DNA in solution are also influenced by the salt concentration [35], it is likely that some salt effects on the daunorubicin-DNA interaction may originate from alteration of DNA structure. This interpretation is in agreement with the binding parameters found for the PM2 closed circular DNA at 0.1 M NaC1 (Table II). An appreciably reduced T A B L E II BINDING PARAMETERS WITH PM2 DNA

FOR

THE INTERACTION

OF DAUNORUBICIN

AND DOXORUBICIN

All e x p e r i m ~ m t s w e r e c a r r i e d o u t a t 2 0 ° C i n 0.O1 M TrisoHC1 ( p H 7 . 0 ) , 0 . 1 M NaCI a n d 0 . 5 m M E D T A . See M a t e r i a l s a n d M e t h o d s a n d l e g e n d t o F i g . 1 for other d e t a i l s . Antibiotic

Daunorubiein Doxorubicln

Closed-circular PM2 DNA

Sonicated PM2 DNA

K a p p (M -1 )

n

K a p p (M -1 )

n

6.7 • 106 (±0.6) 8.8 • 106 (±0.5)

0.130 (±0.004) 0.164 (±0.006)

3.6 • 106 4.9 • l0 G

0.170 0.180

211 TABLE III EFFECT OF SPERMINE ON THE BINDING OF ANTHRACYCLINES

TO CALF THYMUS DNA

All e x p e r i m e n t s w e r e c a r r i e d o u t a t 2 0 ° C i n 0 . 0 1 M T r i s - H C l ( p H 7 . 0 ) , 0 . 0 1 M N a C l , 0 . 5 m M E D T A a n d v a r i o u s c o n c e n t r a t i o n s o f s p e r m i n e , as i n d i c a t e d . See T a b l e I f o r f u r t h e r d e t a i l s . Concn. spermine hydrochloride (M) 0 0 . 5 • 1 0 -5 1 . 0 • 1 0 -5 2.0 • 1 0 -5

Daunorubicin

Doxorubicin

Kap p (M-I)

n

Kap p (M-I)

n

12.2 7.0 4.0 1.3

0.220 0.145 0.132 0.120

19.1 7.5 4.3 2.2

0.212 0.178 0.172 0.150

• • • •

106 106 106 106

• 106 • 106 • 106 • 106

number of binding sites was found only in the case of the daunorubicin-DNA interaction. The supercoiled DNA had a higher affinity for the drug than the corresponding nicked or linear DNA, as shown by the affinity for the sonicated PM2 DNA. A different antibiotic binding affinity of circular compared to linear PM2 DNA is attributed to the free energy associated with the superhelical formation [4,36]. The interpretation that daunorubicin binding is more sensitive to the alteration of DNA structure is supported by binding studies performed in the presence of spermine (Table III). The polyamine competes with antrhacyclines for DNA binding much more efficiently than NaC1, since the effect of 2 . 1 0 - S M spermine is similar to that produced by 1 M NaC1. However, the number of binding sites of daunorubicin is less affected by the presence of spermine than by the alkaline cation. This aliphatic tetramine, which binds preferentially in the minor groove of the DNA double helix [37], does not appreciably alter the DNA structure [38]. The high efficiency of spermine in competing with anthracyclines for binding to DNA does not support models in which the amino sugar lies in the major groove [39]. Indeed, intercalation of daunorubicin into synthetic DNA is not perturbated by bulky substituents at position 5 of the pyrimidine ring, which projects into the major groove of the double helix [40]. Factors other than salts may be critical in establishing the structure of polynucleotides [41]. Changes in the secondary and/or tertiary (supercoiling or packing) structure of DNA may be induced by polypeptides [42,43] or by histones in chromatin [44,45]. Although the conformation of DNA in chromatin is essentially similar to that found for DNA in solutions (B structure) [46], there must be some modification of the B form, since in the nucleosome the DNA is coiled around the histone core [47], i.e., the extent to which the B form is modified depends on the tertiary structure [48]. The results of the binding of daunorubicin and doxorubicin to unsheared calf thymus chromatin (under low ionic strength conditions to avoid aggregation and removal of bound proteins [49]) are shown in Fig. 3. Since the Scatchard plots of the daunorubicin binding to chromatin are not linear (Fig. 3A), it is difficult to evaluate accurate binding parameters. For other inter-

212

B

1 al

0 T

i

\ ~

i

Fig. 3. S e a t c h a r d p l o t o f d a u n o r u b t c i n ( A ) a n d d o x o r u b i e i n (B) b i n d i n g t o c a l f thymus e h r o m a t i n . All e x p e r i m e n t s w e r e c a r r i e d o u t a t 2 0 ° C in 0 . 0 1 M Tris-HC1 ( p H 7 . 0 ) , 0 . 0 1 M NaC1 a n d 0 . 5 m M E D T A . In t h e c a s e o f d a u n o r u b i c i n (A), t h e t w o t y p e s o f b i n d / n g sites, t e n t a t i v e l y d e s c r i b e d b y t h e s t r a i g h t lines, a r e characterized by the following binding parameters: Kapp(1) = 7 • 106 M -I and n I = 0.100; Kapp(2) = 3 • 106 M-I and n 2 = 0.170.

calating agents, this curvature of the binding plots has been interpreted as reflecting a heterogeneity of binding sites in chromatin [7]. At low ionic strength dannorubicin binds to chromatin at two different sets of sites, as previously reported for ethidium bromide [7,9,10]. An extrapolation of the high affinity region to r/c = 0 gives values of n lower than those found for free DNA under these conditions (Table I). The two types of binding sites displayed by chromatin are characterized by binding constants (Fig. 3) similar to those found for the circular and linear DNA, respectively, at higher ionic strength (i.e., at 0.1 M NaCI) (Tables I and II). Distinct curvature in the Scatchard plots has been consistently found for the interaction of chromatin with daunorubicin, but not for the interaction with doxorubicin at least in the range of r below 0.15. Typical curves are presented in Fig. 3B. In this case, it appears that the presence of chromosomal proteins mainly lowers the binding strength ( g a p p ---- 5 " 1 0 6 -+ 0 . 9 M - 1 ) without affecting the number of primary binding sites (n = 0.160 -+ 0.02). The binding constant is consistent with the ionic strength effect expected from the electrostatic interaction of proteins with the DNA phosphate backbone. Indeed, the isotherm for the binding of doxorubicin to ct'.romatin is very similar to that for the binding to DNA at higher ionic strength [16]. In both cases, deviations from non-linearity occur at a high doxorubicin : nucleotide ratio (i.e., at r values above 0.15). This striking difference in the chromatin binding features of the two antibiotics further supports the view that doxorubicin binding is less ser~itive to the changes in DNA conformation than is daunorubicin. Consistent with this interpretation are the different ionic strength effects on the daunorubicin and doxorubicin-DNA interactions. The reasons for the difference in the interaction specificity of daunorub~in and doxorubicin to DNA are not clear at the molecular level. Detailed examination of a modified intercalation model [50] has shown that doxorubicin can participate in an additional hydrogen-bonded

213 interaction, compared to daunorubicin, which involved the C-14 hydroxyl group and the DNA phosphate group at the intercalation site. This additional interaction may be involved in both the different interaction specificity reported in this work and the enhanced DNA binding affinity of doxorubicin compared to daunorubicin. This interpretation is consistent with the suggestion that substituents in ring A of the anthracycline chromophore are necessary for strong DNA binding [19,51]. Therefore, in addition to the amino sugar, it appears that the side chain also plays an important role in determining the strength and the specificity of the interaction. It is unclear at present whether or not these differences at the molecular level contribute to the increased therapeutic effectiveness of doxorubicin as compared to daunorubicin. However, it is of interest to note that, in spite of the marked difference in cellular accumulation (the C-14 hydroxy group decreases the membrane permeability), the nuclear concentrations of the two antibiotics are very similar, thus suggesting an increased nuclear affinity of doxorubicin [52]. Acknowledgements This work was supported in part by research grants Nos. 77.01692.04 and 78.02829-96 from the Consiglio Nazionale delle Ricerche, Rome. We thank Ms. M.A. Rapuano for assistance in preparation of the manuscript. References 1 Goldberg, I.H., Beerman, T.A. and Poon, R. (1977) Cancer (Becket, F.F., ed.), Vol. 5, PP. 427--456, Plenum Press, New York 2 Neidle, S. (1979) Progress in Medicinal Chemistry (Ellis, G.P. a nd West, G.B., eds.), Vol. 16, pp. 151-221, Elsevier/North-HoUand Biomedical Press, A m s t e r d a m 3 Blake, A. and Peacocke, A.R. (1968) BioPolymers 6, 1225--1253 4 Waring, M.J. (1972) The Molecular Basis of Antibiotic A c t i o n (Gale, E.R., Cundiiffe, E., Reynolds, P.E., R i c h m o n d , M.H. and Waring, M.J., eds.), pp. 173--277, J ohn Wiley, L o n d o n 5 Miiller, W. an d Crothers, D.M. (1968) J. Mol. Biol. 35, 251--290 6 Wang, J.C. (1974) J. Mol. Biol. 89, 783--801 7 Angerer, L.M., Gerghiou, S. and Moudrianakis, E.N. (1974) Biochemistry 13, 1 0 7 5 - - 1 0 8 2 8 Lurquin, P.F. (1974) Chem.-Biol. Interact. 8, 303--313 9 Lawrence, J.J. and Daune, M. (1976) Biochemistry 15, 3301--3307 10 Paoletti, J., Magee, B.B. and Magee, P.T. (1977) Biochemistry 16, 351--357 11 Houssier, C., Bontemps, J., Edmonds-Aft, X. and Fredericq, E. (1977) Ann. N.Y. Acad. Sci. 303, 170--189 12 Wartell, R.M., Larson, J.E. and Wells, R.D. (1975) J. Biol. Chem. 250, 2 6 9 8 - - 2 7 0 2 13 Soben, H.M., Reddy, B.S., Bhandary, K.K., Jain, S.C., Sakore, T.D. and Seshadri, T.P. (1978) Cold Spring Harbor Symp. Quant. Biol. 42, 87--102 14 Di Marco, A., Areamone, F. and Zunino, F. (1975) Antibiotics (Corcoran, J.W. and Hahn, F.E., eds.), Vol. 3, pp. 101--128, Sprlnger-Verlag, Berlin 15 Di Marco, A., Casazza, A.M., Gambetta, R., Supino, R. and Zunino, F. (1976) Cancer Res. 36, 1962--1966 16 Zunino, F., Gambetta, R., Di Marco, A., Velcich, A., Zaccara, A., Quadrifoglio, F. and Creszenzi, V. (1977) Biochim. Biophys. Acta 476, 38--46 17 Zunino, F., Gambetta, R., Di Marco, A., Luoni, G. and Zacc~Lra, A. (1976) Biochem. Biophys. Res. Commun. 69, 7 44--750 18 Zunino, F., Di Marco, A. and Velcich, A. (1977) Cancer Lett. 3, 271--275 19 Zunlno, F., Di Marco, A. and Zaceara, A. (1979) Chem.-Biol. Interact. 24, 217--225 20 Byrn, S.R. and Dolch, G.D. (1978) J. Pharm. Sci. 67, 688--693 21 Miiller, W. and Crothers, D.M. (1975) Eux. J. Biochem. 54, 267--277 22 Vig, B.K. (1977) Mutat. Res. 49, 189--238 23 AUfrey, V.G., Mizsky, A.E. and Osawa, S. (1957) J. Gen. Physiol. 40, 4 5 1 - - 4 9 0

214 24 Clark, R.J. and Felsenfeld, G. (1971) Nat. New Biol. 229, 101--106 25 Thomas, C.A., Berns, K.I. and Kelly, T.J. (1967) Procedures in Nucleic Acid Rese~Lrch (Cantoni, G.L. and Davies, D.R., eds.), pp. 535--540, Harper and Row, New Y o r k 26 Espejo, R.T., Canelo, E.S. and Sinsheimer, R.L. (1969) Proc. Natl. Acad. Sci. U.S.A. 63, 1 1 6 4 - - 1 1 6 8 27 Hinnen, R., Schafer, R. and Franklin, R.M. (1974) Eur. J. Biochem. 59, 1--14 28 Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 5 1 , 6 6 0 - - 6 7 2 29 Daune, A.P. (1972) Eur. J. Biochem. 26, 207--211 30 Record, M.T., Jr., Lohman, T.M. and de Haseth, P. (1976) J. Mol. Biol. 107, 145--158 31 Le Pccq, J.B. and Paoletti, C. (1967) J. Mol. Biol. 27, 87--106 32 Huang, Y.M. and Phillips, D.R. (1977) Biophys. Chen. 6, 363--368 33 Festy, B., Sturm, J. and Daune, M. (1975) Biochim. Biophys. Acta 407, 24--42 34 Gaugain, B., Barbet, J., Capelie, N., Roques, B.P. and Le Pecq, J.B. (1978) Biochemistry 17, 5078-5088 35 Hanlon, S., Chan, A. and Berman, S. (1978) Biochim. Biophys. Acta 519, 526--536 36 Bauer, W. and Vinograd, J. (1970) J. Mol. Biol. 47, 4 1 9 - - 4 3 5 37 Liquorl, A.M., Costantino, L., Crescenzi, V., Elia, V., Giglio, E., Puliti, R., De Santis Savino, M. and Vitagliano, V. (1967) J. Mol. Biol. 24, 113--122 38 Waring, M.J. (1970) J. Mol. Biol. 64, 247--279 39 Pigram, W.J., Fuller, W. and Hamilton, L.D. (1972) Nat. New Biol. 235, 17--19 40 Patel, D.J. and Canuel, L.L. (1978) Eur. J. Biochem. 90, 247--254 41 Yon Hippeli, P.H. and McGhee, J.D. (1972) Annu. Rev. Biochem. 41, 231--300 42 Herskovits, T.T. and Brahms, J. (1976) Biopolymers 15, 687--706 43 Weiskopf, M. and Li, H.J. (1977) Biopolymers 16, 669--684 44 Dalgleish, D.G., Dings4yr, E. and Peacocke, A.R. (1973) Biopolymers 12, 445---457 45 Felsenfeld, G. (1978) Nature 2 7 1 , 1 1 5 - - 1 2 2 46 Brain, S. (1971) J. Mol. Biol. 58, 277--288 47 Komberg, R.D. (1977) Annu. Rev. Biochem. 46, 931--954 48 Goodwin, D.C. and Brahms, J. (1978) Nucleic Acids Res. 5, 835--850 49 Li, H.J., Hu, A.W., Maciewicz, R.A., Cohen, p., santella, R.M. and Chang, C. (1977) Nucleic Acids Res. 4, 3 8 3 9 - - 3 8 5 4 50 Neidle, S. and Taylor, G. (1977) Biochim. Biophys. Acta 479, 450---459 51 Henry, D.W. (1976) Cancer Chemotherapy, ACS S y m p o s i u m Series 30 (Sartorelii, A.C., ed.), pp. 15-57, American Chemical Society, Washington 52 NSel, G., Peterson, C., Trouct, A. and Tulkens, P. (1978) Eur. J. Cancer 14, 3 6 3 - - 3 6 8