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. 1972 Jun;128(1):117–132. doi: 10.1042/bj1280117

Methylation of ribonucleic acid by the carcinogens dimethyl sulphate, N-methyl-N-nitrosourea and N-methyl-N′-nitro-N-nitrosoguanidine. Comparisons of chemical analyses at the nucleoside and base levels

P D Lawley 1, S A Shah 1
PMCID: PMC1173576  PMID: 4673570

Abstract

1. The following methods for hydrolysis of methyl-14C-labelled RNA, and for chromatographic isolation and determination of the products, were investigated: enzymic digestion to nucleosides at pH6 or 8; alkaline hydrolysis and conversion into nucleosides; hydrolysis by acid to pyrimidine nucleotides and purine bases, or completely to bases; chromatography on Dowex 50 (NH4+ form) at pH6 or 8.9, or on Dowex 50 (H+ form), or on Sephadex G-10. 2. The suitability of the various methods for determination of methylation products was assessed. The principal product, 7-methylguanosine, was unstable under the conditions used for determinations of nucleosides. 3- and 7-Methyladenine and 3- and 7-methylguanine are best determined as bases; 1-methyladenine and 3-methylcytosine can be isolated as either nucleosides or bases; O6-methylguanine is unstable under the acid hydrolysis conditions used and can be determined as the nucleoside; 3-methyluracil was detected, but may be derived from methylation of the ionized form of uracil. 3. Differences between the patterns of methylation of RNA and homopolyribonucleotides by the N-methyl-N-nitroso compounds and dimethyl sulphate were found: the nitroso compounds were able to methylate O-6 of guanine, were relatively more reactive at N-7 of adenine and probably at N-3 of guanine, but less reactive at N-1 of adenine, N-3 of cytosine and probably at N-3 of uridine. They probably reacted more with the ribose–phosphate chain, but no products from this were identified. 4. The possible influences of these differences on biological action of the methylating agents is discussed. Nitroso compounds may differ principally in their ability to induce miscoding in the Watson–Crick sense by reaction at O-6 of guanine. Both types of agent may induce miscoding to a lesser extent through methylation at N-3 of guanine; both can methylate N atoms, presumably preventing Watson–Crick hydrogen-bonding. N-Methyl-N-nitrosourea can degrade RNA, possibly through phosphotriester formation, but this mechanism is not proven.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abell C. W., Rosini L. A., Ramseur M. R. Alkylation of polyribonucleotides: the biological, physical, and chemical properties of alkylated polyuridylic acid. Proc Natl Acad Sci U S A. 1965 Aug;54(2):608–615. doi: 10.1073/pnas.54.2.608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. BORSOOK H., DEASY C. L., HAAGENSMIT A. J., KEIGHLEY G., LOWY P. H. Incorporation in vitro of labeled amino acids into proteins of rabbit reticulocytes. J Biol Chem. 1952 May;196(2):669–694. [PubMed] [Google Scholar]
  3. Bollack C., Keith G., Ebel J. P. Alcoylation des acides necléiques. I. Etude comparée de la méthylation des acides ribonucléiques en solution aqueuse et organique. Bull Soc Chim Biol (Paris) 1965;47(5):765–785. [PubMed] [Google Scholar]
  4. DRUCKREY H., IVANKOVIC S., PREUSSMANN R. SELEKTIVE ERZEUGUNG MALIGNER TUMOREN IM GEHIRN UND RUECKENMARK VON RATTEN DURCH N-METHYL-N-NITROSOHARNSTOFF. Z Krebsforsch. 1965 Feb 3;66:389–408. [PubMed] [Google Scholar]
  5. Delihas N., Staehelin M. The preparation of rat-liver soluble ribonucleic acid. Biochim Biophys Acta. 1966 May 19;119(2):385–391. doi: 10.1016/0005-2787(66)90196-1. [DOI] [PubMed] [Google Scholar]
  6. Druckrey H., Kruse H., Preussmann R., Ivankovic S., Landschütz C. Cancerogene alkylierende Substanzen. 3. Alkyl-halogenide, -sulfate, -sulfonate und ringgespannte Heterocyclen. Z Krebsforsch. 1970;74(3):241–273. [PubMed] [Google Scholar]
  7. Holy A., Scheit K. H. Die Methylierung von Dinucleosidphosphaten mit Diazomethan. Biochim Biophys Acta. 1967 Apr 18;138(2):230–240. [PubMed] [Google Scholar]
  8. Junowicz E., Spencer J. H. Rapid separation of nucleosides and nucleotides by cation-exchange column chromatography. J Chromatogr. 1969 Oct 28;44(2):342–348. doi: 10.1016/s0021-9673(01)92545-2. [DOI] [PubMed] [Google Scholar]
  9. KIRBY K. S. ISOLATION AND CHARACTERIZATION OF RIBOSOMAL RIBONUCLEIC ACID. Biochem J. 1965 Jul;96:266–269. doi: 10.1042/bj0960266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. KRIEK E., EMMELOT P. METHYLATION AND BREAKDOWN OF MICROSOMAL AND SOLUBLE RIBONUCLEIC ACID FROM RAT LIVER BY DIAZOMETHANE. Biochemistry. 1963 Jul-Aug;2:733–740. doi: 10.1021/bi00904a019. [DOI] [PubMed] [Google Scholar]
  11. LAWLEY P. D., BROOKES P. FURTHER STUDIES ON THE ALKYLATION OF NUCLEIC ACIDS AND THEIR CONSTITUENT NUCLEOTIDES. Biochem J. 1963 Oct;89:127–138. doi: 10.1042/bj0890127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. LUDLUM D. B. ALKYLATION OF POLYNUCLEOTIDE COMPLEXES. Biochim Biophys Acta. 1965 Apr 19;95:674–676. doi: 10.1016/0005-2787(65)90525-3. [DOI] [PubMed] [Google Scholar]
  13. Lawley P. D. Effects of some chemical mutagens and carcinogens on nucleic acids. Prog Nucleic Acid Res Mol Biol. 1966;5:89–131. doi: 10.1016/s0079-6603(08)60232-9. [DOI] [PubMed] [Google Scholar]
  14. Lawley P. D. Methylation of DNA by N-methyl-N-nitrosourethane and N-methyl-N-nitroso-N'-nitroguanidine. Nature. 1968 May 11;218(5141):580–581. doi: 10.1038/218580a0. [DOI] [PubMed] [Google Scholar]
  15. Lawley P. D., Orr D. J., Shah S. A. Reaction of alkylating mutagens and carcinogens with nucleic acids: N-3 of guanine as a site of alkylation by N-methyl-N-nitrosourea and dimethyl sulphate. Chem Biol Interact. 1972 May;4(6):431–434. doi: 10.1016/0009-2797(72)90064-6. [DOI] [PubMed] [Google Scholar]
  16. Lawley P. D., Orr D. J. Specific excision of methylation products from DNA of Escherichia coli treated with N-methyl-N'-nitro-N-nitrosoguanidine. Chem Biol Interact. 1970 Aug;2(2):154–157. doi: 10.1016/0009-2797(70)90047-5. [DOI] [PubMed] [Google Scholar]
  17. Lawley P. D., Thatcher C. J. Methylation of deoxyribonucleic acid in cultured mammalian cells by N-methyl-N'-nitro-N-nitrosoguanidine. The influence of cellular thiol concentrations on the extent of methylation and the 6-oxygen atom of guanine as a site of methylation. Biochem J. 1970 Feb;116(4):693–707. doi: 10.1042/bj1160693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Litwack M. D., Weissmann B. Source of urinary 8-hydroxy-7-methylguanine in man. Biochemistry. 1966 Sep;5(9):3007–3012. doi: 10.1021/bi00873a033. [DOI] [PubMed] [Google Scholar]
  19. Loveless A., Hampton C. L. Inactivation and mutation of coliphage T2 by N-methyl-and N-ethyl-N-nitrosourea. Mutat Res. 1969 Jan-Feb;7(1):1–12. doi: 10.1016/0027-5107(69)90043-8. [DOI] [PubMed] [Google Scholar]
  20. Loveless A. Possible relevance of O-6 alkylation of deoxyguanosine to the mutagenicity and carcinogenicity of nitrosamines and nitrosamides. Nature. 1969 Jul 12;223(5202):206–207. doi: 10.1038/223206a0. [DOI] [PubMed] [Google Scholar]
  21. Ludlum D. B. Alkylated polycytidylic acid templates for RNA polymerase. Biochim Biophys Acta. 1970 Jul 16;213(1):142–148. doi: 10.1016/0005-2787(70)90015-8. [DOI] [PubMed] [Google Scholar]
  22. Ludlum D. B. Chemical methylation of synthetic polynucleotides. Mol Pharmacol. 1966 Nov;2(6):585–592. [PubMed] [Google Scholar]
  23. Ludlum D. B. Ethylation of polyadenylic acid. Biochim Biophys Acta. 1969 Feb 18;174(2):773–775. doi: 10.1016/0005-2787(69)90311-6. [DOI] [PubMed] [Google Scholar]
  24. Ludlum D. B., Wilhelm R. C. Ribonucleic acid polymerase reactions with methylated polycytidylic acid templates. J Biol Chem. 1968 May 25;243(10):2750–2753. [PubMed] [Google Scholar]
  25. MAGEE P. N., FARBER E. Toxic liver injury and carcinogenesis. Methylation of rat-liver nucleic acids by dimethylnitrosamine in vivo. Biochem J. 1962 Apr;83:114–124. doi: 10.1042/bj0830114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Macon J. B., Wolfenden R. 1-Methyladenosine. Dimroth rearrangement and reversible reduction. Biochemistry. 1968 Oct;7(10):3453–3458. doi: 10.1021/bi00850a021. [DOI] [PubMed] [Google Scholar]
  27. Magee P. N., Barnes J. M. Carcinogenic nitroso compounds. Adv Cancer Res. 1967;10:163–246. doi: 10.1016/s0065-230x(08)60079-2. [DOI] [PubMed] [Google Scholar]
  28. Olson A. O., Baird K. M. Single-strand breaks in Escherichia coli DNA caused by treatment with nitrosoguanidine. Biochim Biophys Acta. 1969 Apr 22;179(2):513–514. doi: 10.1016/0005-2787(69)90063-x. [DOI] [PubMed] [Google Scholar]
  29. Pochon F., Michelson A. M. Polynucleotides. IX. Methylation of nucleic acids, homopolynucleotides and complexes. Biochim Biophys Acta. 1967 Nov 21;149(1):99–106. doi: 10.1016/0005-2787(67)90694-6. [DOI] [PubMed] [Google Scholar]
  30. SHUGAR D., FOX J. J. Spectrophotometric studies of nucleic acid derivatives and related compounds as a function of pH. I. Pyrimidines. Biochim Biophys Acta. 1952;9(2):199–218. doi: 10.1016/0006-3002(52)90147-9. [DOI] [PubMed] [Google Scholar]
  31. Shooter K. V., Edwards P. A., Lawley P. D. The action of mono- and di-functional sulphur mustards on the ribonucleic acid-containing bacteriophage mu2. Biochem J. 1971 Dec;125(3):829–840. doi: 10.1042/bj1250829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Singer B., Fraenkel-Conrat H. Chemical modification of viral ribonucleic acid. 8. The chemical and biological effects of methylating agents and nitrosoguanidine on tobacco mosaic virus. Biochemistry. 1969 Aug;8(8):3266–3269. doi: 10.1021/bi00836a020. [DOI] [PubMed] [Google Scholar]
  33. Singer B., Fraenkel-Conrat H. Chemical modification of viral ribonucleic acid. VII. The action of methylating agents and nitrosoguanidine on polynucleotides including tobacco mosaic virus ribonucleic acid. Biochemistry. 1969 Aug;8(8):3260–3266. doi: 10.1021/bi00836a019. [DOI] [PubMed] [Google Scholar]
  34. Smith K. D., Armstrong J. L., McCarthy B. J. The introduction of radioisotopes into RNA by methylation in vitro. Biochim Biophys Acta. 1967 Jul 18;142(2):323–330. doi: 10.1016/0005-2787(67)90615-6. [DOI] [PubMed] [Google Scholar]
  35. Swann P. F., Magee P. N. Nitrosamine-induced carcinogenesis. The alklylation of nucleic acids of the rat by N-methyl-N-nitrosourea, dimethylnitrosamine, dimethyl sulphate and methyl methanesulphonate. Biochem J. 1968 Nov;110(1):39–47. doi: 10.1042/bj1100039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Uziel M., Koh C. K., Cohn W. E. Rapid ion-exchange chromatographic microanalysis of ultraviolet-absorbing materials and its application to nucleosides. Anal Biochem. 1968 Oct 24;25(1):77–98. doi: 10.1016/0003-2697(68)90083-3. [DOI] [PubMed] [Google Scholar]
  37. WATSON J. D., CRICK F. H. Genetical implications of the structure of deoxyribonucleic acid. Nature. 1953 May 30;171(4361):964–967. doi: 10.1038/171964b0. [DOI] [PubMed] [Google Scholar]
  38. Whittle E. D. Methylation of rat-liver RNA in vivo by methyl methanesulphonate. Biochim Biophys Acta. 1969 Dec 16;195(2):381–388. doi: 10.1016/0005-2787(69)90644-3. [DOI] [PubMed] [Google Scholar]
  39. Wolfenden R. V., Kirsch J. F. Enzymatic displacement of oxygen and sulfur from purines. J Am Chem Soc. 1968 Nov 20;90(24):6849–6850. doi: 10.1021/ja01026a054. [DOI] [PubMed] [Google Scholar]
  40. Zoltewicz J. A., Clark D. F., Sharpless T. W., Grahe G. Kinetics and mechanism of the acid-catalyzed hydrolysis of some purine nucleosides. J Am Chem Soc. 1970 Mar 25;92(6):1741–1749. doi: 10.1021/ja00709a055. [DOI] [PubMed] [Google Scholar]

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