Ethylation of polyadenylic acid

773

BIOCHIMICAET BIOPHYSICA ACTA PRELIMINARY

NOTES

BBA 91240

Ethylation

of polyadenylic

acid

Ethylating agents have a somewhat different mutagenic effect from methylating agents in many biological systems. In comparative chemical studies, REINER AND ZAMENHOF~ found several derivatives after calf thymus DNA was reacted with dimethyl sulfate. No identifiable derivatives were obtained when DNA was treated with diethyl sulfate, but protamine titrations indicated that some reaction, interpreted as phosphate esterification, had occurred. LETT, PARKINS AND ALEXANDER~ supported this interpretation with physical data obtained during the reaction of DNA with ethyl methanesulfonate. Following our use of synthetic polynucleotides as models for nucleic acids in similar reactions3, we have treated polyadenylic acid (poly A) with ethyl methanesulfonate. Although the chemistry of this reaction is similar in some respects to the corresponding methylation reaction 3,4, the effects on polymeric structure are entirely different. Poly A of IIO ooo molecular weight was obtained from Miles Chemical Co. Nuclease activity was absent as shown by the light scattering experiments described below. Reagent grade ethyl methanesulfonate and methyl methanesulfonate were purchased from the Eastman Kodak Co.; similar results were obtained with redistilled ethyl methanesulfonate. Although the same reactions occurred more slowly at 37’, alkylations were generally performed at 50” to obtain convenient rates for the study of molecular weight changes. After alkylation, polynucleotides were precipitated quantitatively with ethanol, redissolved in water, dialyzed against 0.15 M NaCl-0.015 M sodium citrate, 0.1 M NaCl, and distilled water, and lyophilized to dryness. To determine composition, the alkylated polymers were hydrolyzed in I M HCl at 100’ for 30 min; adenine was separated from its derivatives by chromatography on Dowex 50 with a pH 6.5 ammonium acetate system. Derivative-containing fractions were collected, evaporated to dryness, and characterized by ultraviolet spectra obtained on a Cary model 15 spectrophotometer, and by paper chromatography on Whatman No. I filter paper. Solvent systems used were: (A) methanol-cont. HCl-water (7 :2 : I, v/v/v) and (B) n-butanol-aq. NH, (p = o.88)-water (85 : z : 12, v/v/v). Authentic I-methyladenine was obtained from Cycle Chemical Corp. for comparison, and r-ethyladenine was prepared from adenosine by a method known to give I substitution5. Light scattering experiments were performed in cacodylate buffer at pH 7 as described previously4 except that a thermostated jacket was used to maintain the temperature of the solutions at 50” in a standard cylindrical cell. Over IO times the concentration of alkylating agent was required to produce an equivalent amount of substitution in poly A with ethyl methanesulfonate as with methyl methanesulfonate. Below 8 O/9substitution, the only detectable derivative in the hydrolyzed polymer was the corresponding I-alkyladenine. Identification of rAbbreviation:

poly

A, polyadenylic

acid. Biochiin.

Biophys.

Acta,

174

(1969) 773-775

methyladenine has been described previously3. The derivative released form ethylated poly A was identical in chromatographic behavior (Solvents A and B) and in ultraviolet spectra to the r-ethyladenine prepared from adenosine5. It had a spectrum which was similar in both acid (A,,, := 260 mp) and base (Amax= 271 m/r) to that of r-methyladenine; the mobilities referred to adenine in Solvent A (1.5) and in Solvent B (1.3) were similar to those given by LAWLEYAND BR~OKW for their presumptive r-ethyladenine; and the pK determined spectrophotometrically (7.0) was similar to that given by PALMfor I-ethyladenine.

fi

oi

i REACTION

TIME(h)

Fig. r. Effect of methylation and ethylation on molecular weight of poly A: ratio of molecular weight to initial molecular weight, Mw/MwO, TJBYSUS time. Reactions were carried out at 50’ in cacodylate buffer (initial pH 7; I 0.2) under the following conditions. Methylation reaction: poly A, 0.8 mg/ml; methyl methanesulfonate, 2.1 mg/ml; final pH 6.8; adenine substituted, 3.9 %. Ethylation reaction (I): poly A, 0.8 mg/ml; ethyl methanesulfonate, 8.5 mg/ml; final pH, 6.6; adenine substituted, less than I %. Ethylation reaction (2): poly A, 0.8 mg/ml; ethyl methanesulfonate, 20.5 mg/ml; final pH, 6.2; adenine substituted, I “0.

The most surprising effect of ethylation is shown in Fig. I, which clearly demonstrates the chain scission that occurs when poly A is treated with ethyl methanesulfonate. Polymer degradation was observed repeatedly under conditions similar to those given in the legend of Fig. I, and followed a similar time course, becoming very slow after approx. 4 h. Ethylated poly A which had been isolated was, itself, stable under these conditions. Since the pH falls somewhat during these reactions, it was necessary to demonstrate that the polymer was stable under these conditions. Control experiments were performed in which the pH was lowered with I M HCl to 6.1, which is below the minimum reached during ethylation. No change in molecular weight was observed for several hours under these conditions. Furthermore, the same chain scission was “observed when ethylation was performed in a more concentrated buffer in which pH was held between 6.8 and 7.1. No such degradation was observed at comparable or higher levels of purine methylation. On the other hand, an aggregation reaction has been observed during methylation at room temperature4. Since it was possible that such aggregation masked concurrent chain scission, the control methylation shown in Fig. I was performed. At the end of 3 h and 3.9 y0 methylation, neither aggregation nor degradation was observed at this temperature; Biochim.

Biophys.

Acta,

‘74

(1969)

773-775

PRELIMINARY

NOTES

775

more importantly, when the pH was increased to IO with NH, at room temperature to dissociate any aggregates which might have been formed, no chain scission was uncovered. Thus, the ability to cause chain scission is possessed by ethyl methanesulfonate but not by methyl methanesulfonate. Although these studies demonstrate a chemical difference between methylation and ethylation, they do not provide any information on the mechanism of chain degradation. However, if a direct attack cn the sugar-phosphate bond is involved, this may reflect a difference in the tendency of ethyl methanesulfonate and methyl methanesulfonate to undergo SN, and SN, reactions. It is probably premature to relate these results directly to the different biological effects of methylation and ethylation, but it is easy to visualize a mechanism whereby chain scission could result in mutations. The skilled technical assistence of Mrs. Charlotte Mao is gratefully acknowledged. The author is a Markle Scholar in Academic Medicine and is supported by Career Development Award I-KLI-GM-19754 from the National Institute of General Medical Sciences. Financial assistance was also received from American Cancer Society grant T-432 and from Public Health Service grant GM 12416. Department of Pharnzacology,

DAVID B. LUDLUM*

University School of Medicine, New Haven, Corn.. (U.S.A.) Yale

I 2 3 4 5 6 7

B. REINERAND S.ZAMENHOF, J.Biol. Chem., 228 (1957)475. J. T. LETT, G. ,M. PARKINS AND P. ALEXANDER, Arch. Biochem. Biophys., 97 (1962) 80. D.B.LuDLuM, R. C.WARNERAND A. J.WAHBA, Science, 145 (1964) 397. D. B. LUDLUM, Biochim. Biophys. Acta, II~ (1966) 630. N. J. LEONARD AND T. FUJII, Proc. hTatl. Acad. Sci. U.S., 51 (1964) 73. P. D. LAWLEY AND P.BROOKES, Biochem. J., 8g (1963) 127. B. C. PAL, Biochemistry, I (1962) 558.

Received November rgth, 1968 ’ Present address: Department of Cell Biology School of Medicine, Baltimore, Md. ZIZOT, U.S.A.

and Pharmacology,

Biochim.

Biophys.

University

Acta,

of Maryland

174 (1969) 773-775