The mode of interaction of actinomycin D with deoxyribonucleic acid

BIOCHIMICA ET BIOPHYSICA ACTA

641

BBA 95053

THE MODE OF INTERACTION OF ACTINOMYCIN D WITH DEOXYRIBONUCLEIC

ACID

L I E B E F. CAVAL1ERI AND R O B E R T G. N E M C H I N

Sloan-Kettering Division, Graduate School o/ Medical Sciences, Cornell University 2VIedical College, New Yorh, N. Y. (U.S.A.) (Received F e b r u a r y 4th, I964)

SUMMARY

The interaction of actinomycin D with DNA from calf thymus and Escherichia coli has been examined by equilibrium dialysis and light scattering. There are two types of binding sites, one of which has a binding constant about 50 times greater than the other. The binding of actinomycin to the strong sites causes a "dimerization" of the DNA. Upon removal of the drug, the molecular weight reverts to its original value. The radius of gyration of the dimerized complex is not sensibly greater than that of the original DNA; therefore the doubling of the molecular weight is due to a lateral rather than a head-to-tail aggregation. Subsequent binding of actinomycin has so little effect on the radius of gyration that intercalation can be ruled out. Denaturation of the DNA results in a single type of binding site, with a binding constant of intermediate value, but the total number of sites remains constant. These observations shed light on the nature of the actinomycin binding sites and their relation to the interaction of RNA polymerase (nucleoside triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6 ) with DNA. It is deduced that actinomycin inhibits RNA polymerase by competing with it for the strong sites on the DNA, and that these sites are in one groove while those that bind DNA polymerase are in the other groove.

INTRODUCTION

Actinomycin D inhibits RNA synthesis both in vivo 1 and in vitro ~-4 by binding to the template DNA. The interaction in vitro has been shown to occur mainly with the guanine residues 5-8. In this paper we present the results of equilibrium dialysis binding studies and light scattering. These two experimental methods are complementary in that the former provides information regarding the interaction of the drug at specific sites while the latter yields knowledge of the size and shape of the actinomycin-DNA complex. The dual approach has permitted a more explicit interpretation of the interaction. It has been found that a small fraction of the total amount of the actinomycin binds very tightly to DNA. It is deduced that the inhibition of RNA polymerase (nucleoside triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6 ) by actinomycin is due to this small fraction rather than to the total amount bound. Biochim. Biophys. Acta, 87 (1964) 641-652

642

L, F. C A V A L I E R I A N D R. G. N E M C H I N

METHODS

Light scattering A Brice-Phoenix light scattering photometer was used for all measurements. The details of the procedure have been described previously 9,1°. The wavelength of the incident beam was 5460 ~-. No correction for absorption due to actinomycin is necessary at this wavelength since the transmittance at 546o A, as measured in a Beckman spectrophotometer, was sufficiently great ( > 93 %) for all concentrations of actinomycin studied. The fluorescence was found to be negligible. Since the drug is bound to DNA, it was necessary to determine the refractive index increment of the complex, using the equation (dn/dC)c°mplex

--

(dn/dC)DNA + B (dn/dC)Act I + B

where B is the weight ratio of actinomycin bound to DNA, calculated directly from the binding data; i.e., the values of r in Table I were converted to a weight-weight TABLE

I

BINDING AND LIGHT SCATTERING RESULTS C a r r i e d o u t w i t h u n d e n a t u r e d D N A a t p H 7-5, 0.2 M NaC1; l i g h t s c a t t e r i n g c a r r i e d o u t a t 24 °. C a l f - t h y m u s D N A c o n c e n t r a t i o n w a s o.13 m g / m l . E , coli D N A c o n c e n t r a t i o n w a s o.12 m g / m l . r is t h e r a t i o of m o l e s of a c t i n o m y c i n b o u n d p e r m o l e of D N A n u c l e o t i d e . T h e e r r o r i n t h e r a d i u s of g y r a t i o n , p, is a b o u t i oo ~ . c is f r e e a c t i n o m y c i n c o n c e n t r a t i o n . 25 °

~lc × ro -3 42.0 40.0 23. 7 7.8 4.8 4.0 3.3

r o I i i i I i

:8o0 :12o :68.1 :38.2 : 3o.6 :26.1

Cal/ t h y m u s

DNA

4°

c(aM)

Mol. wt.(×xo -~)

p(A)

r/c×zo -3

r

c(itM)

o.o 0.030 0.36 1.92 5.3 ° 8.40 11. 3

1.8 2.4 3.1 3.2 -3.5 3-4

18oo 176o 212o 195 ° -194 ° 1945

42.0 23.t, lO. 4 7.0 5.3 3.8 --

o I i i I i

:125 :59 :33 :22. 7 : 20. 4 --

o.o 0.35 1.63 4,3 ° 8.30 12.8 --

1675 -2000 19oo 216o 2240

49.0 21.3 4.5 1. 5 I.O .

o I :83.3 I : 62.5 I :52.8 I :45.5 . .

E . coli D N A

44.0 17.8 6.6 4,1 2.8 2. 3

o I I I I I

:86 : 52 :37.5 :31.7 :27. 5

o.o 0.64 2.96 6.68 11. 7 16.1

1.32 -3. I 3.0 3.1 3.3

.

o.o o.56 3.56 12.7 22.o

basis. As a check, the refractive index increment was determined for a mixture of DNA and actinomycin and found to be in agreement with that calculated for a mixture of complex and free actinomycin. I t is assumed in using this equation that all DNA molecules are equally capable of binding actinomycin. The refractive index increment at 5460 A is 0.208 for DNA and 0.20o for actinomycin. The second virial coefficient, which is usually zero for DNA, was found to have a slight positive value for D N A complexes containing relatively large amounts of actinomycin. Molecular weight determinations were carried out on solutions which Biochim.

Biophys.

A c t a , 87 (1964) 641 652

INTERACTION OF ACTINOMYCIN D WITH D N A

643

had been studied b y equilibrium dialysis (see below), so that the concentration of complex could be determined directly. The excess turbidity over the appropriate solvent was used in the calculations. Scattering due to solvent alone did not vary much for the various actinomycin concentrations.

Equilibrium dialysis 2o ml of DNA solution (about o.I mg/ml) were placed in a Visking casing bag and dialyzed against an equal volume of actinomycin D solution. The solutions were placed in screw-cap vials and rotated about once per sec for 24 h. The temperature was either 25 ~ o . 5 ° or 4:~o.5 °. After this period both the outer and inner fluids were read, obviating the necessity for a correction due to the absorption of actinomycin b y the membrane. Readings were made at 4600 A, which is the isosbestic point for actinomycin-DNA mixtures. Therefore no correction for the effect of DNA on the spectrum of actinomycin was necessary. Dialyses were carried out in 0.2 M NaC1 at approximately the following initial actinomycin concentrations (in ttg/ml): i, io, 25, 50, 75, ioo. The pI-[ of the outer solution was adjusted to the desired value by addition of either I-[C1 or Na2COa. The pI-I values of the inner and outer solutions were determined after equilibration. Tile absorbance of the outer solution represents the free actinomycin concentration, denoted as c in Table I. This value was subtracted from that of the inner solution to give the amount of actinomycin bound. When the latter (in moles/l) is divided by the nucleotide or phosphate concentration (in moles/l), the values for r given in Table I are obtained. The molar extinction coefficient of actinomycin at 4600 A is 18 ooo. Binding was shown to be reversible by equilibrating a previously equilibrated actinomycin-DNA solution against fresh 0.2 M NaC1. The values for r and c so obtained were those expected for the total actinomycin concentration contained in the bag. MATERIALS

Calf-thymus DNA was isolated according to a modification 9 of the procedure of KAY et al. n. The weight average molecular weight was 1. 9. lO 6. Denaturation was accomplished by heating at IOO° for IO rain in 0.2 M NaC1, followed by fast cooling. This treatment did not sensibly alter the molecular weight. I t is of interest that two similar samples of D N A which had been stored as fibers at 4 ° in a desiccator at 75 % relative humidity for 2 years increased in molecular weight to 3" lO6. On heating to IOO° the weight decreased to 0.5. lO 6. Thus not only did aggregation occur on standing in the solid state in the cold, but also nuclease action. Escherichia coli B DNA was isolated as described previouslyg, TM. The weight average molecular weight was 1. 4. lO 6. Actinomycin D was a gift from Dr. L. SARETT, Merck, Sharpe & Dohme, Rahway, N. J. RESULTS AND DISCUSSION

Equilibrium dialysis Theory: In simple binding theory, if it is assumed that the DNA has n sites with binding constant K, then the following equation holds: r/c = Kn--Kr (I) Biochim. Biophys. Acta, 87 (1964) 641-652

644

L.F.

C A V A L I E R I AND R. G. N E M C H I N

where r refers to the n u m b e r of sites per mole of nucleotides occupied at a free concentration c of actinomycin. A plot of r/c vs. r yields a straight line whose ordinate intercept is K n and whose abscissa intercept is n. Thus K can be explicitly calculated. W h e n there is more than one type of binding site the simple Eqn. I does not hold and must be replaced b y a more complex expression. If the nucleic acid has two types of sites, n 1 and n2, with binding constants K 1 and K S respectively, then Eqn. 2 m a y be applied. Klnl

K.n2

Here n l + n 2 = n, the total n u m b e r of sites (per mole of nueleotides). The four unknowns can be evaluated in the manner previously described la. Clearly there m a y be more than two types of sites, b u t the data can be accounted for with this minimum number N u m b e r o/ sites and binding constants: Fig. I shows a binding curve of calft h y m u s D N A at 25 °, p H 7.5. Since the curve is not linear, we can conclude t h a t there are at least two types of sites. The broken line is a theoretical curve calculated to fit the experimental curve from the values of the parameters nl, n2, K~, and K S (Table II). Tile strong-binding sites (K S = 74.1o a) attract actinomycin with about 50 times the strength of the weak group of sites. There are about IO times as m a n y sites in the 4L

4 4 4

3 ] 3 3

2 ?

¢x ' 2 t.

1

1 l ] ]

0

0.Ol

0.0Z

0.113

0.04

O.0~

0.00

0.07

t"

F i g . I. B i n d i n g of a c t i n o m y c i n t o c a l f - t h y m u s D N A (o.13 m g / m l ) . T h e NaC1 c o n c e n t r a t i o n w a s 0.2 M i n a l l c a s e s . ® , 4 ° , p H 7.5; © , 25 ° , p H 7,5; O , 25 ° , p H 3.5; A , 4 ° , p H 2.7; ~ , 25 ° , p H 7.5 ( d e n a t u r e d ) . T h e b r o k e n l i n e i s a t h e o r e t i c a l c u r v e for b i n d i n g a t 25 °, p H 7.5, c a l c u l a t e d as i n dicated in the text. The other theoretical curves have been omitted to avoid crowding the figure.

l~iochim. Biophys. Acta, 87 (1964) 6 4 1 - 6 5 2

INTERACTION OF ACTINOMYCIN D

645

WITH D N A

latter group (n t = o.o55 ) as in the former (n2 = 0.005). The biological significance of this will be discussed later. TABLE II BINDING CONSTANTS FOR CALF-THYMUS AND E . coli D N A B i n d i n g s t u d i e s c a r r i e d o u t i n 0.2 M NaC1. The b i n d i n g c o n s t a n t s K 1 a n d K 2 c o r r e s p o n d t o t h e sites nl a n d n~, r e s p e c t i v e l y . F o r d e n a t u r e d D N A t h e r e is o n l y one v a l u e for t h e b i n d i n g c o n s t a n t K a n d t h e n u m b e r of sites n since r/c vs. r is a l i n e a r re l a t i on, Sample

Calf Calf Calf Calf

K x xo -~

K1 × xo -5

1. 4 2. 3

Kz X zo - 5

n

nl

ne

74.o 64.0

o.o55 0.060

o.oo5 0.005

0.030 0.02

O.OLO O.OLO

25 4 25 4

1.6 1.5

o.o6o 0.065 0.054 0.020

Calf t h y m u s ( d e n a t u r e d )

7.5

25

6.0

0.063

(native) (native)

7.5 7.5

25 4

coli cull

(native) (native) (native) (n ative)

Temp.

7.5 7.5 3.5 2. 7

E. E.

thymus thymus thymus thymus

pH

2.3 7.5

38.0 40.0

0.040 0.024

The binding of actinomycin to E. cull DNA is somewhat different (Fig. 2; Tables I and II). Here we note t h a t there are significantly fewer weak sites (nl o.o3), while the strong sites are greater in n u m b e r (n 2 = o.0I). Also, the difference between the binding constants K 1 and K s is n o t a b l y less than for calf-thymus DNA. 5O 48 46 42 40 38

36 N

32 30

28 26 7

24 22 r_ 18 16 12 10

0

0.01

0,02

0.03

0.04

r Fig. 2. B i n d i n g of a c t i n o m y c i n to E . cull D N A (o.12 m g / m l ) i n 0.2 M NaC1, p H 7.5. O, 25°; ®, 4 °. Biochim.

Biophys.

Acta,

87 (1964) 641-652

646

L.F.

C A V A L I E R I A N D R. G. N E M C H I N

647

These DNA's cannot be compared rigorously because of the long extrapolation to the ordinate intercept; but it is nevertheless safe to conclude that the ratio nl/n 2 is different for the two. The difference m a y reside not only in base composition and sequence, but possibly in a fundamental difference in structure. Such a difference has been demonstrated in previous studies la, in which, for example, it was found that the degradation of the molecule to smaller fragments requires more phosphate ester cleavages in the case of E. coli DNA than for calf-thymus DNA. This indicated that the former possessed more strands than the latter. E[/ect o/pH: The effect of p H was examined by carrying out equilibrium dialyses at two lower p H values: 3.5 and 2. 7 (Table I H ) . At both p H values the binding curve TABLE

IIl

BINDING OF ACTINOMYCIN TO CALF-THYMUS D N A

(pH

3.5, 0.2 M NaC1, 25°.)

rlc × xo - 3

7.2 6. 4 3.2 2.6

AS A FUNCTION OF p H

I I I I

r

c(taM)

: 133 :57.2 :31.3 : 27

1.o 4 2.73 IO.O 14.2

p H 2. 7, 4 °. 2.0 o. 9 0.9

I : 133 I :80 I :62.5

3.75 13. 9 17.8

is a straight line (Fig. I). This shows that only one type of site is involved. The binding constants are similar to that for the weak-binding sites at p H 7.5 (Table II). At p H 3.5 only the high-binding sites have been eliminated (n = 0.054; compare to nl = 0.055 at p H 7.5, Table II), but at p H 2.7 m a n y of the weak-binding sites also have been eliminated (n = 0.o2). I t is significant that at pt{ 2. 7 the binding constant is nearly the same as that at pt{ 3.5 (the small difference m a y be due to the fact that binding at p H 2.7 was carried out at 4 ° to prevent denaturation). The constancy of the binding constant corroborates the assumption that there is only one type of site in the weak group. That no irreversible change was caused b y lowering the p H to 2.7 was shown by reneutra]izing the DNA-actinomycin mixture to p H 7.5 and observing that the amount bound was identical to that of the original mixture at pt{ 7.5To determine whether the alteration of the pI-[ affected the DNA or the actinomycin, both the spectrum and the electrophoretic mobility of actinomycin were investigated as a function of pH. There were no significant spectral changes between p H 3 and IO. Such changes might be expected if there were a titratable group on the ring. There was no electrophoretic mobility on paper between p}{ 2.7 and IO, other than that due to electroendosmosis. On this basis we conclude that there is no charge on the actinomycin molecule in this entire p H range. The fact that a change in ionic strength does not alter the binding, as shown by spectral shifts n, suggests that the interaction between actinomycin and DNA is not electrostatic. This is consistent with the evidence that actinomycin is uncharged. The change in binding with decreasing p H is most easily attributed to the acquisition of positive charges by certain DNA Biochim.

Biophys.

Acta,

87 (1964) 6 4 1 - 6 5 2

INTERACTION

OF

ACTINOMYCIN

D

WITH

647

DNA

bases, which might affect hydrogen bonding or other interaction with actinomycin. E//ect o/ denaturation: Heat-denatured DNA is remarkably different from native DNA in its binding characteristics (Fig. I, Table ~V). The straight line in Fig. I indicates that there is only one type of site, whose binding constant is 6.0. lO 5. TABLE

IV

B I N D I N G RESULTS WITH DENATURED CALF-THYMUS O N A ( p F I 7.5, 0 . 2 M N a C 1 ,

r/c × i o - a 38.o 32.o 22.8 12.8 6. 3 3.8

25°.)

r

o I i I i I

c(I~M)

MoI. wl. × i o - 6

o.o o.297 I.O 7 3.4 ° 8.27 20. 5

: lO 5 : 41 :23 : 19.2 : 18. 5

1.9 -3.5 --3.7

This value is intermediate between the two binding constants in native DNA. It is noteworthy that the total number of sites is the same (n = 0.063) after denaturation. Therefore the helical structure plays no role in determining the number of sites, but it does affect the strength of binding at those sites. Thermodynamic results: The thermodynamic functions for actinomycin binding to native calf-thymus and E. coli DNA are contained in Table V. Two points are TABLE

V

THERMODYNAMIC RESULTS

T h e r e s u l t s a r e for n a t i v e

DNA

in b o t h cases.

Call t h y m u s D N A 4°

d F1 ° (kcal) A Fz ° (kcal) ,dill° (kcal) dH2°(kcal) AS~ ° e.u. AS2 ° e.u.

E. coli D N A 25 °

-- 6.82 -- 8.66 --3.91 1.o8

25 °

4°

-- 7.04 -- 9.4 ° --3.91 i.io

-- 7.48 -- 7.66 --9.42 --3.20

-- 7.34 -- 9.oo --lO.4 --4.2

lO.5 35.2

--7.0 16.I

worth noting. The standard-state free energies are about the same as those found for rosaniline binding J3. This is of interest since rosaniline bears a permanent positive charge, and binding is due both to electrostatic and Van der Waals forces. Actinomycin D, being uncharged, can bind only non-electrostatically. PEACOCKEAND S K E R R E T T 15 have shown that the loss of the possibility for Van der Waals contact strikingly decreases the binding of acridine derivatives to DNA. Thus 5-aminoacridine, which contains a flat aromatic ring, is strongly bound, while 5-aminotetrahydroacridine, which is puckered, is weakly bound despite the fact that both compounds are positively charged. The second point concerns the entropy changes. The change at the strongbinding sites is considerably more positive than that at the weak sites. This greater disordering effect is most simply interpreted as a displacement of water molecules from the guanine moieties by interaction with the (flat) aromatic ring of the acBiochim.

Biophys.

Acla,

87

(1964)

641-652

(i4~

L. F. C A V A L I E R I AND R. G. NEMCHIN

tinomycin. Such an interaction would probably permit the maximum amount oi water to be displaced from deoxyguanosine, which may have as many as five water molecules associated with iO 6. This implies that at the weak sites the guanine and actinomyein rings do not come completely in contact with each other. Rather, only the "edges" of each ring system may interact (perhaps by the hydrogen bond formarion suggested by HAMILTONet al.~7), with the conccmitant displacement of, roughly, three molecules of water. These suggestions are subject to two limitations. First, we have not considered the possible displacement of water from actinomycin. In addition, bound ions could also be displaced from neighboring groups, such as the phosphate. Secondly, there is uncertainty in the evaluation of the binding constants, because of extrapolation to the ordinate, and therefore in the AF ° values. Qualitatively, however, the relative entropy changes should be valid.

Light scattering The presence of small amounts of actinomycin results in an increase in the molecular weight of both calf-thymus and E. coli DNA (Table I). The increase is considerably greater than that expected to result from the presence of the bound actinomycin, and must be attributed to actinomycin-mediated aggregation of the DNA. The molecular weight is approximately doubled; this may be related to the fact that actinomycin possesses two peptide side chains, each of which might interact with a different DNA molecule. The molecular weight increase parallels strikingly the saturation of the strongbinding (r2) sites (Fig. 3). At concentrations of actinomycin above I.IO -s M the 3.5

®

-v'~-

,o

x

~=

3.0 2.~

0

~

J

3

I

4

0.06 0.04

-----O---"---

0.018" 0.016 0.014 0.012 0.010 0.008 0.006

q

0.004 0.002

~0

20 3O

c x 106 (Free Actinomycin D concn.)

Fig. 3. T o p : M o l e c u l a r w e i g h t of c a l f - t h y m u s D N A as a f u n c t i o n of free a c t i n o m y c i n c o n c e n t r a t i o n . B o t t o m : r 1 a n d r 2 a s a f u n c t i o n of f r e e a c t i n o m y c i n c o n c e n t r a t i o n ; r 1 a n d r , a r e t h e n u m b e r of m o l e s of a c t i n o m y c i n b o u n d p e r m o l e of D N A n u c l e o t i d e for t h e w e a k a n d s t r o n g sites, r e s p e c t i v e l y .

Biochi~n. Biophys. Acta, 8 7 (1964) 6 4 1 - 6 5 2

INTERACTION OF ACTINOMYCIN D WITH DNA

649

increase in molecular weight is due only to the additionally bound actinomycin (r1 sites). The increase in radius of gyration of the aggregated DNA (Table I) is relatively small, suggesting that the "dimerization" occurs in a side-to-side rather than head-to-tail fashion. The aggregation of both calf-thymus and E. coli DNA is reversible, since removal of the actinomycin by shaking with chloroform-octanol restores the original molecular weight. The situation with regard to molecular weight is essentially the same for denatured DNA. Helical structure is therefore not necessary for dimerization. The increase in molecular weight of denatured and undenatured DNA occurs when roughly the same number of actinomycin molecules (about I per IOO nucleotides) has been bound (Table IV). This indicates that dimerization is not specifically mediated by the binding of actinomycin at strong sites, since those sites cannot be distinguished by actinomycin in denatured DNA. Originally, we had intended to use the light scattering method to study changes in the radius of gyration, in order to test whether actinomycin is bound by intercalation in the manner suggested by LERMANis. The increase in molecular weight of the complex has made a simple answer to this question impossible, at least at low actinomycin concentration, where the increase in radius of gyration (p) (from about 18oo • to about 2000 )t,) is greater than could be accounted for by intercalation. At higher actinomycin concentrations, where there is no further aggregation (Table I), the near-constancy of the radius of gyration for calf-thymus DNA suggests that the actinomycin is not intercalated between hydrogen-bonded base pairs. There could however be intercalation in a "hole" created by a base pair which had broken hydrogen bonds and whose bases had rotated into the grooves of the helix. Such intercalation, of course, would not sensibly alter the length of the helix.

CONCLUSIONS AND BIOLOGICAL IMPLICATIONS We have shown that actinomycin binds to at least two types of sites on the calfthymus DNA molecule. The strong-binding (n2) sites comprise about IO ~o of the total and are saturated at low actinomycin concentrations (lower part of Fig. 3). Since there is a sharp, concomitant decrease in the thymus-DNA-primed activity of RNA polymerase at low actinomycin concentrations 19, we deduce that binding at these sites and not at the weak-binding (nl) sites is inhibitory (Fig. 4)- The inhibition may be due either to direct competition between actinomycin and polymerase for the n 2 sites, or to a blocking of specific polymerase sites by the "dimerization" of the template DNA which accompanies actinomycin binding at n2 sites (Figs. 3 and 4). This dimerization, or approximate doubling of molecular weight, has been observed to occur reversibly with both thymus and E. coli DNA. Denatured DNA, on the other hand, has only one type of actinomycin binding site, of intermediate binding constant, although the total number of sites is unchanged. Inhibition of RNA polymerase in this case follows the total binding curve (middle curve of Fig. 5), as expected. However, the dimerization of denatured DNA still occurs at low actinomycin concentration (Table IV). This means that dimerization of denatured DNA is virtually complete while inhibition of its :RNA template activity continues to increase. We conclude then that the doubling of the molecular weight is either not involved at all in the inhibition, or only partially so. Thus, in the case Biochim. Biophys. Acta, 87 (1964) 641-652

650

L.F.

CAVALIERI AND R. G. NEMCHIN

100 o , - _ ~, .....

Weak sites

~ 0

® .~. 7 0 - \ \ ' ,

E D 50 -

a.

30

~ "~

/Vlolecularweight

30L

~

~- Lu ~-

P01ymerase

~: ~

2

11o

........

J

470

" , , ~ ~ _

"

80 ~

10

90

o L

~

0

~

0.002

0.004

l

1

J

0.006

0.008

0.01

,/xL

__i

~

0.03

loo

0.04

moles Actinomycin B/moles BNA nucleotide

Fig. 4. The d a t a for r x and r, f r o m Fig. 3 are replotted (as s t r o n g and w e a k sites) and c o m p a r e d to the p o l y m e r a s e curve of KAHAN et al. t°. All curves except molecular weight curve refer to left ordinate. The inhibition of R N A p o l y m e r a s e parallels r a t h e r closely the increase in molecular weight and the s a t u r a t i o n of the s t r o n g sites. lOO(

-_= 70 =

60 50 40

=

30 29 10 l

2

3

4

5

6

7

8

c x 106 IFree Actin0mycin O c0ncn.t Fig. 5. A c o m p a r i s o n of s t r o n g (rz) a n d w e a k (rl) sites for native c a l f - t h y m u s D N A with those for d e n a t u r e d D N A (r). I n h i b i t i o n of R N A polymerase b y a c t i n o m y c i n follows r 2 curve for native D N A and r curve for d e n a t u r e d DNA. (The ordinate is equivalent to per cent R N A - p o l y m e r a s e a c t i v i t y remaining.)

of native DNA, inhibition by actinomycin is probably also due to a direct competition with RNA polymerase for the strong-binding (n2) sites. The fact that more actinomycin is required to produce the same extent of inhibition in denatured DNA 19 simply means that the binding constant of ~ N A polymerase to D N A increases or remains constant on denaturation while that of actinomycin decreases at those sites. I t is interesting to note that dimerization of both native and denatured D N A occurs after approximately the same amount of actinomycin has been bound. Thus the physical state of the DNA is not important to dimerization. If the n2 sites constitute polymerase binding sites, then it is important to learn their nature since they presumably determine the starting points for RNA synthesis. Since the frequency of the n2 sites is about I per IOO nucleotide pairs, we can deduce, Biochim. 13iophys. Acta, 87 (I964) 641-652

INTERACTION OF ACTINOMYCIN ~D WITH

DNA

651

on the assumption that starting and stopping points are identical (i.e., that RNA synthesis cannot proceed past a strong site), that roughly 65 400 is the average molecular weight of a messenger RNA molecule. Since messenger RN2k is probably at least IO times larger, the hypothesis must be modified. It is clear that RNA polymerase would have to be more selective than actinomycin, requiring additional specificity such as a cluster of n2 sites. Or, the n 2 sites may include two sub-groups, only one of which binds polymerase while both bind actinomycin equally well. In attempting to identify the actinomycin binding sites, it has been shown s that there is a correlation between the amount of actinomycin bound and the guanine content of the DNA. There are several indications that the correlation is not simple, however. First, as demonstrated above, there are at least two types of binding sites. Secondly, the synthetic polymer deG plus deG binds much less actinomycin than expected 10. Finally, the naturally occurring crab testis deAT polymer, which contains only 2.5 % guanine, produces a substantial spectral shift with actinomycin, while synthetic poly deAT produces none s. There can be little doubt that both guanine and its neighboring nucleotides influence the binding. The fact that only about one-third of the guanines in calf-thymus DNA can interact with actinomycin at saturation is probably due both to steric hindrance by the peptide chains of actinomycin and to a requirement for a particular nucleotide sequence. Since the total number of sites remains essentially constant after denaturation of the DN&, the helical structure can be eliminated as a qualitative factor in determining the binding sites. Our data do show, however, that the distinction between strong- and weakbinding sites depends on the secondary structure of the DNA. Thus the same base sequence is probably characteristic of both types of sites, but the affinity for actinomycin appears related to the extent to which the bases are available for interaction with the drug. Since weak-binding sites constitute 9° ~o of the sites found in undenatured thymus DNA, they probably form part of the helical structure. Their interaction with actinomycin may well be through the peripheral formation of hydrogen bonds, as suggested by HAMILTON et al. ~7, on the basis of X-ray crystallographic analysis. The other io ~o of sites, which are strong-binding, may then consist of non-hydrogen-bonded guanine rings which have rotated from the center of the helix into one of the grooves, where they can come into close, two-dimensional contact with the planar ring system of actinomycin. These suggestions are corroborated by the fact that all binding sites are identical in denatured DNA, where all bases are equally available. It therefore appears that inhibition of the RNA.-polymerase reaction results, ultimately, from the planar interaction of the aromatic portions of actinomycin D with certain DNP~ bases. Curiously enough, the DNA-polymerase (EC 2.7.7.7) reaction is only slightly affected thereby, whereas just the reverse is true for proflavine 2. Yet the proflavine molecule is quite similar to the aromatic portion of actinomycin. Tile distinction must be attributed to the long polypeptide side chains of actinomycin, and to some fundamental difference in the interactions of the RNA and DNA polymerases with DNA. The difference may be that RNA polymerase binds in one groove while DNA polymerase binds in the other, as suggested earlier 2o.

Biochim. Biophys. Acta, 87 (1964) 641-652

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L. F. CAVALIERI AND R, G, NEMCHIN ACKNOWLEDGEMENTS

This work was supported in part by funds from the National Cancer Institute, National Institutes of Health, Public Health Service (Grant CY-319o), and the Atomic Energy Commission (Contract No. AT(3o-l)-9IO). REFERENCES 1 j . M. KIRK, Biochim. Biophys. Acla, 42 (196o) 167. 2 j . HURWlTZ, J. J. FORTH, M. MALAMY AND M. ALEXANDER, Proe. Natl. Acad. Sci. U.S., 48 (1962) 1222. a I. H. GOLDBERG AND M. RABINO\VlTZ, Science, 136 (1962) 315. a. E. REICH, I. H. GOLDBERG AND M. RABINOWITZ, Nature, 196 (1962) 7435 H. M. RAUEN, U. KERSTEN AND \V, I~ERSTEN, Z. Physiol. Chem., 321 (196o) 139. 6 W. KERSTEN AND H. KERSTEN, Z. Physiol. Chem., 330 (1962) 21. "vV. KERSTEN, Biochim. Biophys. Acta, 47 ( I 9 6 I ) 61o. 8 I. H. GOLDBERG, M. RABINOWITZ AND E. REICH, Proc. Natl. Acad. Sci. U.S., 48 (I962) 2094. 9 L. F. CAVALIERI, J. DEUTSCH AND B. H. ROSENBERG, Biophys. J., i ( i 9 6 i ) 302. 10 L. F. CAVALIERI, M. ROSOFF AND B. H. ROSI~;NBERG, J. Am. Chem. Soc., 78 (I956) 5239 . 11 E. R. M. KAY, N, SIMMONS AND A. L. DOI'NCE, J. Am. Chem. Soc., 74 (1952) 1724. 12 L. F. CAVALIERI, t3. H. I~OSENBERG AND J. DEUTSCH, Biochem, Biophys. Res. Commun., i (1959) 124. la L. F. CAVALIERI AND A. ANGELOS, J. A ~ . Chem. Soc., 72 (195 o) 4686. 14 L. F . CAVALIERI AND B. I-t. ROSENBERG, Biophys. J., 1 (1961) 317 . 1~ A. R. PEACOCKE AND J. ~ . SKERRETT, Trans. Faraday Soc., 52 (1956) 261. 16 M. FALK, K. A. HARTMAN, Jr. AND R. C. LORD, J. ,4m. Chem, Soc., 85 (1963) 387 . 17 L. D. HAMILTON, W, FULLER AND E. REICIt, Nature, 198 (1963) 538 . is L. S. LERMAN, J. Mol. Biol., 3 (1961 ) 18. 19 E. KAttAN, F. M. KAItAN AND J. HURWITZ, J. Biol. Chen~., 238 (1963) 2491. 2o L. F. CAVALIERI AND B. 1-I. ROSENBERG, IN J. ~ . DAVIDSON AND G. E. COFIN, Progress in Nucleic Acid Research, Vol. 2, A c a d e m i c Press, N e w Y ork, 1963, p. 9.

Biochim. Biophys. Acla, 87 (1964) 641-652