A Study of Methylated Polyadenylic Acid

Abstract

Copolymers with different contents of adenine and N,-methyl adenine residues have been studied, polyadenylic acid being methylated by dimethylsulfate. Thermal stability, circular dichroism and light scattering measurements are in favor of a two-strand structure a t neutral pH. Thermal stability of these products is much less sensitive to ionic strength than that of most polynucleotides, and in some cases independent of ionic strength. These copolymers interact or not with poly(U), depending on the percentage of N,-methyl adenine residues. Copolymers with high contents of methyladenine interact with poly(A) at neutral pH. These copolymers do not bind DNA-dependent RNA polymerase. Some results a t acid pH are also reported.A large number of studies have been devoted to solutions of synthetic polynucleotides in order to understand their physical properties and also because they may be considered as model molecules for deoxyribonucleic acid (DNA). We are interested, in the laboratory, by the properties of chemically methylated DNA [1,2]. Alkylating reagents react with DNA and give rise to physical and biological changes (see reviews [3-51. On the other hand, methylated bases are largely found in natural nucleic acids and their functions are still obscure. For these reasons and in order to explain some results that we found on methylated DNA [2], studies have been carried out with samples of polyadenylic acid, poly(A), which had reacted with dimethyl sulfate. Despite the fact that sites of the reaction are different in native DNA and poly(A), some interesting comparisons are possible [ 21. MATERIAL AND METHODSPolyadenylic Acid Polyadenylic acid was purchased from Miles Chemical Company. After several treatments with phenol, the product was fractionnated in 3 fractions according to the method of Eisenberg and Felsenfeld [6]. Each fraction was dialysed against 0.01 M EDTA, then exhaustively against distilled water and lyophilized. The first fraction was used in this work. MethylationWe used the method of Michelson and GrunbergManago Enzyme. DNA-dependent RNA polymerase (EC 2.7.7.6).(4 mg/ml) were added 0.16 ml tri-n-butylamine and 0.05 ml dimethyl sulfate; the solution was shaken a t 4" for different times (from 5min to 48 h, the amount of methylated adenine residues depends upon the time of reaction). At the termination of the reaction 0.05 ml M cacodylate buffer pH 7 was added; the solution was treated with ether (3 times), dialysed against 0.1 M NaC1, 0.05 M sodium cacodylate, then against distilled water and lyophilized.Methylation occurs in the Nl position of adenine, as shown by identification in the acid hydrolysate (1 N HC1, 100" for 1 h). The hydrolysate was chromatographed in the solvent system n-butanol-NH,OH 6 N (180 : 30, v/v) and only two spots were observed (adenine and Nl-methyl adenine) [8]; in the solvent system isopropanol -concentrated HC1-H,O (170 :41: 39, v/v/v) adenine and Nl-methyl adenine have the same mobility [9], and no other spot was found. The relative percentage of adenine and Nl-methyl a...

Hypothetical Double-Helical Poly(A) Formation in a Cell and Its Possible Biological Significance

“…For example, at room temperature it occurs at pH » 6.0 (0.15 M KCl) and pH » 6.8 (0.001 M KCl) (4). In partially methylated poly(A) (at the N1 position of adenine) this transition takes place at pH 9.1 (0.15 M NaCl) at room temperature (5).…”

Double helical forms of poly (A): possible involvement in biological processes

“…But methylation of riboses [10] or bases [11,12] in polyadenylic acid results in a shift in рК ц of formation of double-stranded helices towards alkaline region. The most significant shift occurs as a result of adenine methylation in the position Nl [11]. In this case poly (A) double-stranded helices are formed in 0.15 M NaCl at pH -9.1.…”