in Deoxyribonucleic

Chemical ERIC HOLWITT’ Department

of Biochemistry,

Acid on

Ethylation AND



College of Physicians and Surgeons, Columbia New York, New York 10032 Received



9, 1974

Ethylation of DNA by diethyl sulfate gave 7-ethylguanine as the major product. Dimethyl sulfate was much more reactive than diethyl sulfate in forming 7-alkylguanine. The hydrodynamic properties of DNA did not change as a direct consequence of ethylation. On incubation at 37”C, the viscosity of ethylated DNA decreased at a rate similar to that of methylated DNA. The rate of depurination of 7-ethylguanine from ethylated DNA was the same as that of 7-methylguanine from methylated DNA. These results demonstrate that ethyl groups have identical effects as methyl groups on the secondary structure and stability of DNA.

In an earlier investigation (l), this laboratory elucidated the macromolecular changes brought about in the structure of DNA on methylating the bases in vitro with chemical methylating agents. Though there was no immediate change in the hydrodynamic properties of DNA on methylation, methylated DNA underwent slow degradation owing to depurination of methylated purines followed by singlestrand cleavage at the apurinic sites and eventual double-strand cleavage. Methylation per se produced few alterations in the secondary structure of DNA. In this context it is of interest to ascertain the effect of ethyl groups on the structure and stability of DNA. Lett et al. (2) reported that DNA treated with ethyl methanesulfonate underwent no changes in structure compared to DNA treated with methyl methanesulfonate, and concluded that ethylating agents alkylated the phosphate groups and not the bases. On the other hand, Bautz and Freese (3), Lawley and Brookes (4), and Rhaese and Freese (5) reported that ethyl methanesulfonate alkylated the bases and

suggested that ethylated DNA depurinated slower than methylated DNA. Strauss and Hill (6) studied the stability of DNA alkylated with dimethyl sulfate, diethyl sulfate, methyl methanesulfonate, and ethyl methanesulfonate. They measured the rate of degradation of ethylated and methylated 32P-labeled DNA to acid-soluble materials on heating or treatment with alkali in order to determine the rate of formation of apurinic sites and single-strand cleavage, respectively. Methylated DNA depurinated 1.3 times faster than ethylated DNA and both formed single-strand breaks at the same rate. This report compares the secondary structure and stability of DNA alkylated to the same extent with diethyl sulfate or dimethyl sulfate. EXPERIMENTAL Diethyl sulfate was obtained from Fischer Scientific Company and distilled before use. [“Cldiethyl sulfate was obtained from New England Nuclear. 7-Ethylguanine was purchased from Cycle Chemical Corporation. All scintillation counting was done in Aquasol solution supplied by New England Nuclear. The preparation of calf thymus DNA and determination of its concentration, viscosity, and T, were carried out as previously described (1). DNA was dissolved in a buffer containing 0.01 M cacodylic acid, 0.07 M sodium cacodylate, 0.001 M Na2EDTA, and

’ Eric Holwitt held a traineeship from the National Institutes of Health (5TOlGMOO255). 161 Copyright 0 1975b:y Academic Press, Inc. All rights of reproduction in any form reserved.




0.128 M NaClO,, pH 7.0. Ethylation required a much higher concentration of alkylating agent than methylation and the buffer in which the DNA was dissolved was not sufficient to neutralize all the acid produced in the reaction (7). Additional sodium cacodylate was added to the reactions to maintain the pH above 6. In all reactions, 25 ml of a DNA solution (1 mg/ml) in cacodylate buffer was mixed with an equal volume of sodium cacodylate solution, diethyl sulfate added, and the mixture shaken at 37°C for 6-10 h. When the diethyl sulfate concentration was 0.16 M, the sodium cacodylate added was 1 M, reaction time 6 h, initial pH 8.0, and final pH 6.6. For 0.4 M diethyl sulfate, the corresponding values were; 1.2 M sodium cacodylate, reaction time 8 h, initial pH 8.0, final pH 6.1. For 0.69 M diethyl sulfate, the values were; 2 M sodium cacodylate, reaction time 10 h, initial pH 8.2, final pH 6.0. At the end of the reaction, the volume of each solution was brought to 250 ml and sufficient sodium cacodylate, ethyl monosulfate (from hydrolyzed diethyl sulfate), and water were added so that the final pH was 6.0, sodium concentration 0.02 M, and DNA concentration 0.1 mg/ml. A separate control was run for each reaction in which DNA was added after the diethyl sulfate was first hydrolyzed overnight and then treated exactly as described above. These solutions were used for all hydrodynamic and optical studies. To determine the extent of ethylation, nature of products, and rate of depurination, reactions similar to those described were run with [“Cldiethyl sulfate in l-ml vol and dialyzed to remove radioactive products. The solutions were finally adjusted to 0.02 M Na+, pH 6.0. The hydrolysis of DNA, analysis of bases by high-voltage electropboresis, and measurement of depurination of labeled ethylated bases have been described previously (1, 8). RESULTS

Products of DNA ethylation. Hydrolysis of ethylated DNA by formic acid and separation of the bases by high-voltage paper electrophoresis at pH 3.25 (1) revealed two new bases. One had the same mobility and uv spectra as authentic 7ethylguanine. The mobility of 7-ethylguanine was the same as 7-methylguanine. When ethylation was carried out with [“Cldiethyl sulfate, the 7-ethylguanine spot contained 80% of the radioactivity of the ethylated DNA. The second new base contained 20% of the starting radioactivity and its mobility was the same as 3methyladenine. The second new base was probably 3-ethyladenine. The main difference between dimethyl

sulfate and diethyl sulfate is in their reactivity towards nucleophiles. Dimethyl sulfate hydrolysis had a half-life at 25°C of 60 min, and at 37°C of 12 min. The corresponding values for diethyl sulfate were 75 min at 25”C, and 24 min at 37°C. Dimethyl sulfate reacted with the nucleophile, thiosulfate, with a half-life of 0.2 min at 25”C, whereas diethyl sulfate had a half-life of 13.0 min at 25°C. Though the corresponding methanesulfonates reacted slower than the sulfates, the methyl derivative reacted 17 times faster than the ethyl derivative with thiosulfate, and 1.7 times faster with water. Table I compares the reactivity of dimethyl sulfate and diethyl sulfate with DNA with respect to formation of 7-alkylguanine. It is clear that dimethyl sulfate is much more reactive than diethyl sulfate and that a higher concentration of the latter reagent is required to give the same extent of alkylation as dimethyl sulfate (1, 4, 5). Experiments which compare the effect of methylating and ethylating agents at equimolar concentration are not very informative unless the extent of reaction with DNA is known. Immediate effects of ethylation. As was the case with methylated DNA (l), ethylated DNA showed no significant loss in viscosity immediately after alkylation. The melting curves for ethylated DNA were as sharp as for the control DNA but were shifted to lower temperatures, the T,,, decrease being greater as the extent of TABLE




0.16 M Diethyl sulfate 0.40 M Diethyl sulfate 0.69 M Diethyl sulfate 0.021 M Dimethyl sulfate 0.21 M Dimethyl sulfate “The reagent in the methyl sulfate

%7-alkylguanine 11 25 40 15 54

conditions for alkylation with radioactive and determination of radioactivity are given Experimental section. The reaction with disulfate was complete in 1 h while with diethyl the reaction required 6-10 h.




ethylation was increased. The hyperchromicities of the samples did not change on alkylation. Changes after ethylation. Although ethylation per se produced few alterations in the secondary structure of DNA, prolonged incubation of ethylated DNA produced profound structural changes. Figure 1 shows the loss in viscosity for DNA ethylated to different extents. It is clear that ethyl groups destabilize DNA structure and lead to degradation. The halflives for the viscosity loss were 72 h, 32 h, and 12 h, for DNA ethylated 11, 25, and 40%, respectively. The half-lives for the viscosity loss of methylated DNA (1) were, 280 h, 50 h, and 21 h for DNA methylated 1, 10, and 30%, respectively. The rates of viscosity loss are very similar for ethylated and methylated DNA and suggest that the same process occurs with both types of DNA. For methylated DNA, low-angle light scattering showed (1) that the viscosity loss was clue to double-strand cleavage and not to denaturation or other conformational changes. The double-strand cleavage is undoubtedly the end result of depurination of 7-ethylguanine followed by single-strand cleavage at apurinic sites. To measure depurination of 7-ethylguanine, the rate of release of this base from radioactive ethylated DNA was determined at 37°C and 70°C. The results are shown in Fig. 2. The first-order rate constant at 37°C was 12.4 x 10e5 min-‘, and at 7O”C, 6.1 x lo-’ min-‘. From these values the energy of activation for depurination of 7-ethylguanine from DNA is 26 kcal mole-‘. The corresponding first-order rate constants for depurination of 7methylguanine from DNA (1) were 8.5 x lo-’ min-’ at 37”C, and 8.2 x 10m3min-’ at 70°C. The energy of activation for this reaction was 30 kcal mole-‘. It is clear that the rates of depurination of 7-ethylguanine and 7-methylguanine are the same. DISCUSSION

When the effect of ethylating and methylating agents are compared, not with respect to molarity of reagent, but with respect to degree of alkylation of guanine, it is found that both reagents cause the

FIG. 1. Loss in viscosity of ethylated DNA. DNA ethylated to the extents indicated were incubated at pH 6.0 in cacodylate buffer at 37°C and viscosities measured at intervals. “%q remaining” is the ratio of the specific viscosity at each time interval to the specific viscosity at t = 0, multiplied by 100. Control DNA lost no viscosity over the same time interval.

200 0 200




0 CPm 100

I/I: I/I:







cpm 200 100 r





FIG. 2. Release of 7-ethylguanine from ethylated DNA. DNA ethylated 40% with radioactive ethyl groups was incubated under the same conditions as in Fig. 1 at 37°C and 70°C. Samples were removed at intervals, subjected to high-voltage paper electrophoresis, and radioactivity of the 7-ethylguanine determined.

same changes in the structure of DNA. With both alkylating agents, similar products are formed and there are little immediate changes in the hydrodynamic properties of the alkylated DNA. On incubation, ethylated and methylated DNA undergo depurination of 7-alkylguanine at similar rates and lose viscosity, which is due to double-strand scission, at similar rates. These results establish conclusively that ethyl and methyl groups have identical




effects on the structure of DNA and that the mechanism of degradation is the same in both cases. In uitro, alkylation of DNA leads to loss of transforming activity and at equal concentrations of alkylating agents, methylation is more effective than ethylation (9, 10). The magnitude of the relative effects of methylating and ethylating agents on transforming DNA is the same as the chemical reactivity of the two reagents toward alkylation of the guanine residues in DNA (4, 5). In view of the similar stabilities of ethylated and methylated DNA and the greater reactivity of methylating agents, it is likely that the loss in transforming activity of alkylated DNA proceeds by the same mechanism whether the alkyl groups are methyl or ethyl groups.

REFERENCES 1. UHLENHOPP, E. L., AND KRASNA, A. I. (1971) Biochemistry 10.3290. 2. LETT, J. T., PARKINS, G. M., AND ALEXANDER, P. (1962) Arch. Biochem. Biophys. 97, 80. 3. BAUTZ, C., AND FREESE, E. (1960) Proc. Nat. Acad. Sci. USA 46, 1585. 4. LAWLEY, P. D., AND BROOKES, P. (1963) Biochem. J. 89, 127. 5. RHAESE, H. J., AND FREESE, E. (1969) Biochim. Biophys. Acta 190, 418. 6. STRAUSS, B., AND HILL, T. (1970) Biochim. Biophys. Acta 213, 14. 1. UHLENHOPP, E. L., AND KRASNA, A. I. (1969) Nature (London) 223, 1267. a. TRIFUNAC, N. P., AND KRASNA, A. I. (1974) Biochemistry 13, 2403. 30, 89. 9. STRAUSS,B. S. (1963) J. Gen. Microbial. 10. RHAFSE, H. J., AND BOETKER, N. K. (1973) Eur. J. Biochem. 32, 166.

Structural alterations in deoxyribonucleic acid on chemical ethylation.

ARCHIVES OF BIOCHEMISTBY AND Structural 167,161-164 BIOPHYSICS Alterations (1975) in Deoxyribonucleic Chemical ERIC HOLWITT’ Department of B...
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