Biochimica et Biophysica Acta, 335 (197~) 49-53 ~) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

BBA 97883

ON THE POSSIBILITY OF INTERCALATION OF AROMATIC AMINO ACID

RESIDUES INTO DOUBLE-STRANDED DNA HELIX

JOHN JACOBSEN and JAMES C. WANG

Department of Chemistry, University of California, Berkeley, Calif. 94720 (U.S.A.)

(Received September 19th, 1973)

SUMMARY

The relative sedimentation rates of linear and covalently closed 2 DNA in sucrose gradients containing varying amounts of tryptamine, tyramine, histamine and phenethylamine were measured. The presence of the aromatic amines does not sig- nificantly change the sedimentation coefficient of the closed DNA, indicating that the DNA helix rotation angle is not significantly altered. This lack of angular al- teration is also supported by sedimentation measurements of 2 DNA which had been covalently closed by ligase in the presence of tryptamine. These results suggest that intercalation of aromatic amino acid residues into DNA is unlikely, contrary to the conclusion drawn from other measurements.

INTRODUCTION

The mechanisms of specific interactions between proteins and double-stranded DNA are of much chemical and biological interest (for a review, see ref. 1). The va- rious mechanisms proposed to date can be divided roughly into three categories: (1) The binding involves little or no change of the secondary structure of the DNA helix. The recognition of a unique sequence of bases is achieved by interactions between the protein molecule and groups in the grooves of the DNA helix. (2) Unwinding of the DNA strands occurs at a specific site, to give a single-stranded region or a unique protrusion (such as a hairpinned structure on each strand) for the binding site of the protein. (3) Specific groups on the protein can intercalate into the DNA helix, and can, therefore, interact with specific base sequences.

The last possibility was raised recently by a number of studies on interactions between nucleic acids and amino acids and oligopeptides [2-6]. Results obtained from fluorescence, proton magnetic resonance, circular dichroism and viscosity measurements are suggestive that intercalation of the aromatic residues of amino acids and oligopeptides into the DNA helix might occur.

In cases where intercalative interaction is believed to occur, it is generally found that an unwinding of the DNA helix results [7-9]. Therefore, measurements on the angular alteration of the DNA helix by the binding of aromatic residuals of

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amino acids should be informative in examining the possibility of intercalative in- teraction. We report here that no appreciable angular alteration of the DNA helix is detectable upon binding of tryptamine, tyramine, histamine, and phenethylamine.

MATERIALS AND METHODS

3H- and 14C-labelled 2 phage were obtained by thermal induction of Esche- riehia coli 159 T - (2ci857S7). Purification of phage, extraction of DNA, and prepara- tion of covalently closed D N A were done as described previously [10]. To prepare circular 2 D N A containing approx, one random single-chain scission per molecule, covalently closed 2 D N A in a medium containing 10 mM Tris-HCl, pH 8, 2 mM MgClz, 1 mM Na3EDTA, and 80 #g/ml of bovine plasma albumin was treated with pancreatic DNase (Worthington) at 0 °C. With 0.08#g/ml of DNase, approx. 10 min are needed to introduce one nick per DNA molecule on the average. The enzyme reaction has a high activation energy and reproducible results were obtained only if the temperature was maintained at 0 °C during mixing of the enzyme and the D N A solutions as well as during incubation. The reaction was stopped by addition of 1/20 vol. 0.2 M Na3EDTA and extracting the mixture three times with phenol. After dialyzing out the phenol, the D N A was banded in a CsCl-ethidium bromide gradient to isolate the nicked species from unreacted closed circles [11]. Ethidium was re- moved by n-butanol extraction as described previously [10].

Two methods were used in the determination of the angular alteration of the D N A helix by aromatic amines. In one set of experiments, 3H-labelled covalently closed 2 D N A (obtained by ligase treatment of nicked 2 circles at 30 °C as described in ref. 10) and tgC-labelled linear 2 D N A were sedimented at 4 °C through 5-20 % sucrose gradients containing l mM sodium cacodylate, pH 7, l m M NaCl, and varying amounts of amines. After 160 min at 40 000 rev./min in a Beckman Model L2-65B ultracentrifuge with an SW 50.1 rotor, the gradients were dripped and counted as described previously [12]. For each gradient, the ratio R of the sedimentation rates of the 3H-labelled closed circles and the 14C-labelled linear marker were ob- tained from the ratio of the distances sedimented by the two species [13]. The sedi- mentation medium was chosen to reproduce closely the conditions at which binding measurements were carried out by H616ne and coworkers [2-4]. In the absence of the amines, R is approx. 1.35 and each closed 2 DNA molecule contains approx. 15 negative superhelical turns (or a superhelical density [14] of approx. - 0.003) under the sedimentation conditions. At this degree of superhelicity, R is a sensitive function of the number of superhelical turns [15]. Therefore, a change in R would occur if the addition of an amine causes an unwinding of the DNA helix. It is known that the number of superhelical turns of a covalently closed DNA, and therefore R, is dependent on the temperature [15]. To avoid variation in R due to change in temperature at which sedimentation was carried out, six gradients containing vary- ing amounts of an amine were usually centrifuged simultaneously in a single run. The ratio r of the sedimentation coefficient of the covalently closed D N A in the pres- ence of an amine at concentration c, to that of the same DNA in the absence of the amine measured in the same run, is obtained from r = R(c)/R(O), where R(c) and R(0) are the R values at amine concentrations c and 0, respectively.

In a second series of measurements, 3H-labelled 2ci857S7 D N A containing

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approx, one deoxyribonuclease-introduced nick was converted to the covalently closed form by E. coli ligase at 30 °C in 10 mM Tris-HCl, pH 8, 2 mM MgC12, 1 mM NaaEDTA, and 0.2 or 1.5 mM of an amine. 14C-labelled 2ci857S7 DNA, also lightly nicked by pancreatic deoxyribonuclease, was converted to the covalently closed form by ligase in the same medium except that no amine was added. The two samples were mixed after closure by ligase, phenol extracted, and dialyzed exhausti- vely against 0.1 M NaC1, 0.01 M NaaEDTA. The mixture was then sedimented on a sucrose gradient containing 1 mM NaCI, 1 mM sodium cacodylate, pH 7. I f the binding of a ligand causes of a change of the helix rotation angle, then the removal of the ligand from the 3H-labelled D N A after closure by ligase would give a D N A with different number of twists compared with the 14C-labelled DNA, which was covalently closed in the absence of the ligand but under otherwise identical condi- tions [9, 12]. This difference in the number of twists would cause a difference in sedi- mentation coefficients.

R E S U L T S A N D D I S C U S S I O N

Results obtained from sedimentation runs in media containing 1 mM sodium cacodylate, pH 7, 1 mM NaCI and varying amounts of the aromatic amines trypta- mine, phenethylamine, tyramine, and histamine are tabulated in Table I.

T A B L E I

R E L A T I V E S E D I M E N T A T I O N R A T E O F C O V A L E N T L Y C L O S E D 2 D N A A S A F U N C T I O N

O F T H E C O N C E N T R A T I O N O F A R O M A T I C A M I N E P R E S E N T I N T H E S E D I M E N T A T I O N

M E D I U M

C o n c e n t r a t i o n

o f a m i n e ( M ) T r y p t a m i n e P h e n e t h y l a m i n e T y r a m i n e H i s t a m i n e

0 1.00 1.00 1.00 1.00

2" 10 - s - - 0.95 0.98

1" 10 - 4 1.05 0.97 - -

3 " 10 - 4 1.04 - - -

5 - 10 - 4 1.01 0.99 - -

7 " l 0 - 4 0.99 0.97 - -

1 • l 0 - a 0.98 0.97 0.99 0.99

The error in r in these measurements is estimated to be 4-0.03. Thus it ap- pears that the addition of amines does not change r significantly. Since the amines were added as the hydrochlorides, there was a change in the ionic strength of the sedimentation medium upon their addition. To test whether this change in ionic strength might affect r, a control experiment was done in which NaCI instead of an amine was added to the gradients. The r values obtained for gradients containing 0, 3 • 10 -4, 5 • 10 -4, 7 • 10 -4, and 2 • 10 -a M NaC1 were 1.00, 1.04, 1.01, 1.00, and 0.97, respectively. The small variations are comparable to variations observed with the amines but our data are not of sufficient accuracy to determine whether these variations are significant.

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For tyramine at room temperature in 1 mM sodium cacodylate, pH 7, I mM NaCI, H616ne, et al. [3] found that its binding to D N A followed the Langmuir iso- therm with a binding constant K of approx. 103. M -1 and a maximal number of sites n of approx. 0.2 per D N A phosphate. Therefore at a tyramine concentrat ion o f 10 -3 M, approx. 0. I site per D N A phosphate or one half of all potential sites are occupied. If each tyramine unwinds the D N A by q9 degree, the number of negative super- helical turns introduced into the D N A molecule by the binding of 0.1 N tyramine molecules would be 0.1 N ~p/360, where N z 9.3 • 10 4 is the number of phosphates per 2 D N A [16]. For a 2 D N A with a degree of superhelicity in the range of approx. 10-40 negative superhelical turns per D N A , r changes by 0.013 when the number o f superhelical turns is changed by 1 [12]. Since the observed change in r in our ex- periment is less than 0.06 (which is twice the experimental error), the change in the number of superhelical turns is less than 5. I f 0.1 N ~p/360 is less than 5, (p is less than 0.2 degree. This estimate shows that little unwinding of the D N A helix is occurring upon binding of a tyramine molecule. The binding constant for t ryptamine is of the same order of magnitude as tyramine and the same conclusion holds. For histamine and phenethylamine, no quantitative binding data are available, but the lack of un- winding of the D N A helix in the presence o f these compounds was also observed.

Experiments in which the D N A was covalently closed in the presence of tryp- tamine as described in Materials and Methods also support the conclusion that little or no unwinding of the D N A helix occurs upon binding of the aromatic amine. No detectable change in the helix rotat ion angle was observed with the presence of as much as 1.5 • 10 -3 M tryptamine in the ligase closure medium.

There has been at least one model-building study in the literature indicating that intercalation could occur without a concomitant unwinding of the D N A helix [17]. However, in each case where intercalative type binding is evident, an unwinding of the D N A helix has been observed [7-9]. The smallest unwinding angle reported so far is 4 degrees for daunomycin [8]. The lack of unwinding of the D N A helix upon binding of the aromatic amines is, therefore, indicative that intercalation o f the aro- matic residues into the D N A helix is unlikely, contrary to the conlusion drawn from other measurements.

ACKNOWLEDGEMENT

This research has been supported by grants f rom the U.S. Public Health Ser- vice (GM14621) and the National Science Founda t ion (GB 29331).

REFERENCES

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