DNA and RNA Pyrimidine Nucleobase Alkylation at the Carbon-5 Position

  • Yuri MotorinEmail author
  • Salifu Seidu-Larry
  • Mark HelmEmail author
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 945)


The carbon 5 of pyrimidine nucleobases is a privileged position in terms of nucleoside modification in both DNA and RNA. The simplest modification of uridine at this position is methylation leading to thymine. Thymine is an integral part of the standard nucleobase repertoire of DNA that is synthesized at the nucleotide level. However, it also occurs in RNA, where it is synthesized posttranscriptionally at the polynucleotide level. The cytidine analogue 5-methylcytidine also occurs in both DNA and RNA, but is introduced at the polynucleotide level in both cases. The same applies to a plethora of additional derivatives found in nature, resulting either from a direct modification of the 5-position by electrophiles or by further derivatization of the 5-methylpyrimidines. Here, we review the structural diversity of these modified bases, the variety of cofactors that serve as carbon donors, and the common principles shared by enzymatic mechanisms generating them.


Michael Addition Pyrimidine Ring Enzymatic Mechanism Hydride Transfer Pyrimidine Nucleoside 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. Agrawal N, Lesley SA, Kuhn P, Kohen A. Mechanistic studies of a flavin-dependent thymidylate synthase. Biochemistry. 2004;43(32):10295–301. doi: 10.1021/bi0490439.CrossRefPubMedGoogle Scholar
  2. Auxilien S, Rasmussen A, Rose S, Brochier-Armanet C, Husson C, Fourmy D, et al. Specificity shifts in the rRNA and tRNA nucleotide targets of archaeal and bacterial m5U methyltransferases. RNA. 2011;17(1):45–53. doi: 10.1261/rna.2323411.CrossRefPubMedPubMedCentralGoogle Scholar
  3. Becker HF, Motorin Y, Sissler M, Florentz C, Grosjean H. Major identity determinants for enzymatic formation of ribothymidine and pseudouridine in the T psi-loop of yeast tRNAs. J Mol Biol. 1997;274(4):505–18. doi: 10.1006/jmbi.1997.1417.CrossRefPubMedGoogle Scholar
  4. Bogdanovic O, Gomez-Skarmeta JL. Embryonic DNA methylation: insights from the genomics era. Brief Funct Genomics. 2014;13(2):121–30. doi: 10.1093/bfgp/elt039.CrossRefPubMedGoogle Scholar
  5. Bujnicki JM, Feder M, Ayres CL, Redman KL. Sequence-structure-function studies of tRNA:m5C methyltransferase Trm4p and its relationship to DNA:m5C and RNA:m5U methyltransferases. Nucleic Acids Res. 2004;32(8):2453–63. doi: 10.1093/nar/gkh564.CrossRefPubMedPubMedCentralGoogle Scholar
  6. Burgess AL, David R, Searle IR. Conservation of tRNA and rRNA 5-methylcytosine in the kingdom Plantae. BMC Plant Biol. 2015;15:199. doi: 10.1186/s12870-015-0580-8.CrossRefPubMedPubMedCentralGoogle Scholar
  7. Carreras CW, Santi DV. The catalytic mechanism and structure of thymidylate synthase. Annu Rev Biochem. 1995;64:721–62. doi: 10.1146/ Scholar
  8. Constantinesco F, Motorin Y, Grosjean H. Transfer RNA modification enzymes from Pyrococcus furiosus: detection of the enzymatic activities in vitro. Nucleic Acids Res. 1999;27(5):1308–15.CrossRefPubMedPubMedCentralGoogle Scholar
  9. Conte MR, Galeone A, Avizonis D, Hsu VL, Mayol L, Kearns DR. Solid phase synthesis of 5-hydroxymethyluracil containing DNA. Bioorg Med Chem Lett. 1992;2:79–81.Google Scholar
  10. Edelheit S, Schwartz S, Mumbach MR, Wurtzel O, Sorek R. Transcriptome-wide mapping of 5-methylcytidine RNA modifications in bacteria, archaea, and yeast reveals m5C within archaeal mRNAs. PLoS Genet. 2013;9(6):e1003602. doi: 10.1371/journal.pgen.1003602.CrossRefPubMedPubMedCentralGoogle Scholar
  11. El Yacoubi B, Bailly M, de Crecy-Lagard V. Biosynthesis and function of posttranscriptional modifications of transfer RNAs. Annu Rev Genet. 2012;46:69–95. doi: 10.1146/annurev-genet-110711-155641.CrossRefPubMedGoogle Scholar
  12. Frommer M, McDonald LE, Millar DS, Collis CM, Watt F, Grigg GW, et al. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci U S A. 1992;89(5):1827–31.CrossRefPubMedPubMedCentralGoogle Scholar
  13. Fu L, Guerrero CR, Zhong N, Amato NJ, Liu Y, Liu S, et al. Tet-mediated formation of 5-hydroxymethylcytosine in RNA. J Am Chem Soc. 2014;136(33):11582–5. doi: 10.1021/ja505305z.CrossRefPubMedPubMedCentralGoogle Scholar
  14. Giege R, Helm M, Florentz C. Chemical and enzymatic Probing of RNA Structure. Comprehensive Natural Product Chemistry. In: Söll D, Nishimura S (editors). Oxford: Pergamon Press; 1999;6:63–77.Google Scholar
  15. Goll MG, Kirpekar F, Maggert KA, Yoder JA, Hsieh CL, Zhang X, et al. Methylation of tRNAAsp by the DNA methyltransferase homolog Dnmt2. Science. 2006;311(5759):395–8. doi: 10.1126/science.1120976.CrossRefPubMedGoogle Scholar
  16. Gommers-Ampt JH, Borst P. Hypermodified bases in DNA. FASEB J Off Publ Fed Am Soc Exp Biol. 1995;9(11):1034–42.Google Scholar
  17. Graziani S, Bernauer J, Skouloubris S, Graille M, Zhou CZ, Marchand C, et al. Catalytic mechanism and structure of viral flavin-dependent thymidylate synthase ThyX. J Biol Chem. 2006;281(33):24048–57. doi: 10.1074/jbc.M600745200.CrossRefPubMedGoogle Scholar
  18. Greenberg R, Dudock B. Isolation and characterization of m5U-methyltransferase from Escherichia coli. J Biol Chem. 1980;255(17):8296–302.PubMedGoogle Scholar
  19. Gu X, Ofengand J, Santi DV. In vitro methylation of Escherichia coli 16S rRNA by tRNA (m5U54)-methyltransferase. Biochemistry. 1994;33(8):2255–61.CrossRefPubMedGoogle Scholar
  20. Hamdane D, Argentini M, Cornu D, Golinelli-Pimpaneau B, Fontecave M. FAD/folate-dependent tRNA methyltransferase: flavin as a new methyl-transfer agent. J Am Chem Soc. 2012;134(48):19739–45. doi: 10.1021/ja308145p.CrossRefPubMedGoogle Scholar
  21. Hamdane D, Bruch E, Un S, Field M, Fontecave M. Activation of a unique flavin-dependent tRNA-methylating agent. Biochemistry. 2013;52(49):8949–56. doi: 10.1021/bi4013879.CrossRefPubMedGoogle Scholar
  22. He YF, Li BZ, Li Z, Liu P, Wang Y, Tang Q, et al. Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science. 2011;333(6047):1303–7. doi: 10.1126/science.1210944.CrossRefPubMedPubMedCentralGoogle Scholar
  23. Helm M, Alfonzo JD. Posttranscriptional RNA Modifications: playing metabolic games in a cell’s chemical Legoland. Chem Biol. 2014;21(2):174–85. doi: 10.1016/j.chembiol.2013.10.015.CrossRefPubMedGoogle Scholar
  24. Hong B, Maley F, Kohen A. Role of Y94 in proton and hydride transfers catalyzed by thymidylate synthase. Biochemistry. 2007;46(49):14188–97. doi: 10.1021/bi701363s.CrossRefPubMedPubMedCentralGoogle Scholar
  25. Hussain S, Aleksic J, Blanco S, Dietmann S, Frye M. Characterizing 5-methylcytosine in the mammalian epitranscriptome. Genome Biol. 2013;14(11):215. doi: 10.1186/gb4143.CrossRefPubMedPubMedCentralGoogle Scholar
  26. Islam Z, Strutzenberg TS, Gurevic I, Kohen A. Concerted versus stepwise mechanism in thymidylate synthase. J Am Chem Soc. 2014;136(28):9850–3. doi: 10.1021/ja504341g.CrossRefPubMedPubMedCentralGoogle Scholar
  27. Jurkowski TP, Meusburger M, Phalke S, Helm M, Nellen W, Reuter G, et al. Human DNMT2 methylates tRNA(Asp) molecules using a DNA methyltransferase-like catalytic mechanism. RNA. 2008;14(8):1663–70. doi: 10.1261/rna.970408.CrossRefPubMedPubMedCentralGoogle Scholar
  28. Kealey JT, Santi DV. Identification of the catalytic nucleophile of tRNA (m5U54)methyltransferase. Biochemistry. 1991;30(40):9724–8.CrossRefPubMedGoogle Scholar
  29. Kealey JT, Lee S, Floss HG, Santi DV. Stereochemistry of methyl transfer catalyzed by tRNA (m5U54)-methyltransferase--evidence for a single displacement mechanism. Nucleic Acids Res. 1991;19(23):6465–8.CrossRefPubMedPubMedCentralGoogle Scholar
  30. Kealey JT, Gu X, Santi DV. Enzymatic mechanism of tRNA (m5U54)methyltransferase. Biochimie. 1994;76(12):1133–42.CrossRefPubMedGoogle Scholar
  31. Khursid M, Khan A, Qudrat-E-Khuda M. Synthesis of 5-Hydroxymethylcytidine. J Chem Soc Pak. 1982;4(3):167–8.Google Scholar
  32. Kim J, Xiao H, Bonanno JB, Kalyanaraman C, Brown S, Tang X, et al. Structure-guided discovery of the metabolite carboxy-SAM that modulates tRNA function. Nature. 2013;498(7452):123–6. doi: 10.1038/nature12180.CrossRefPubMedPubMedCentralGoogle Scholar
  33. Kim J, Xiao H, Koh J, Wang Y, Bonanno JB, Thomas K, et al. Determinants of the CmoB carboxymethyl transferase utilized for selective tRNA wobble modification. Nucleic Acids Res. 2015;43(9):4602–13. doi: 10.1093/nar/gkv206.CrossRefPubMedPubMedCentralGoogle Scholar
  34. Koehn EM, Kohen A. Flavin-dependent thymidylate synthase: a novel pathway towards thymine. Arch Biochem Biophys. 2010;493(1):96–102. doi: 10.1016/ Scholar
  35. Koehn EM, Fleischmann T, Conrad JA, Palfey BA, Lesley SA, Mathews II, et al. An unusual mechanism of thymidylate biosynthesis in organisms containing the thyX gene. Nature. 2009;458(7240):919–23. doi: 10.1038/nature07973.CrossRefPubMedPubMedCentralGoogle Scholar
  36. Kong LQ, Zhao J, Fan L, Yang DC. The facile synthetic method of 5-hydroxymethyluracil. Chin Chem Lett. 2009;20(3):314–6.CrossRefGoogle Scholar
  37. Kriaucionis S, Heintz N. The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science. 2009;324(5929):929–30. doi: 10.1126/science.1169786.CrossRefPubMedPubMedCentralGoogle Scholar
  38. Kun A, Szilagyi A, Konnyu B, Boza G, Zachar I, Szathmary E. The dynamics of the RNA world: insights and challenges. Ann N Y Acad Sci. 2015;1341:75–95. doi: 10.1111/nyas.12700.CrossRefPubMedGoogle Scholar
  39. Lartigue C, Lebaudy A, Blanchard A, El Yacoubi B, Rose S, Grosjean H, et al. The flavoprotein Mcap0476 (RlmFO) catalyzes m5U1939 modification in Mycoplasma capricolum 23S rRNA. Nucleic Acids Res. 2014;42(12):8073–82. doi: 10.1093/nar/gku518.CrossRefPubMedPubMedCentralGoogle Scholar
  40. Lindstrom PH, Stuber D, Bjork GR. Genetic organization and transcription from the gene (trmA) responsible for synthesis of tRNA (uracil-5)-methyltransferase by Escherichia coli. J Bacteriol. 1985;164(3):1117–23.PubMedPubMedCentralGoogle Scholar
  41. Machnicka MA, Milanowska K, Osman Oglou O, Purta E, Kurkowska M, Olchowik A, et al. MODOMICS: a database of RNA modification pathways--2013 update. Nucleic Acids Res. 2013;41(Database issue):D262–7. doi: 10.1093/nar/gks1007.CrossRefPubMedGoogle Scholar
  42. Militello KT, Chen LM, Ackerman SE, Mandarano AH, Valentine EL. A map of 5-methylcytosine residues in Trypanosoma brucei tRNA revealed by sodium bisulfite sequencing. Mol Biochem Parasitol. 2014;193(2):122–6. doi: 10.1016/j.molbiopara.2013.12.003.CrossRefPubMedPubMedCentralGoogle Scholar
  43. Mishanina TV, Koehn EM, Conrad JA, Palfey BA, Lesley SA, Kohen A. Trapping of an intermediate in the reaction catalyzed by flavin-dependent thymidylate synthase. J Am Chem Soc. 2012;134(9):4442–8. doi: 10.1021/ja2120822.CrossRefPubMedPubMedCentralGoogle Scholar
  44. Mishanina TV, Corcoran JM, Kohen A. Substrate activation in flavin-dependent thymidylate synthase. J Am Chem Soc. 2014;136(30):10597–600. doi: 10.1021/ja506108b.CrossRefPubMedPubMedCentralGoogle Scholar
  45. Moriya J, Yokogawa T, Wakita K, Ueda T, Nishikawa K, Crain PF, et al. A novel modified nucleoside found at the first position of the anticodon of methionine tRNA from bovine liver mitochondria. Biochemistry. 1994;33(8):2234–9.CrossRefPubMedGoogle Scholar
  46. Motorin Y, Helm M. tRNA stabilization by modified nucleotides. Biochemistry. 2010;49(24):4934–44. doi: 10.1021/bi100408z.CrossRefPubMedGoogle Scholar
  47. Motorin Y, Helm M. RNA nucleotide methylation. Wiley Interdiscip Rev RNA. 2011;2(5):611–31. doi: 10.1002/wrna.79.CrossRefPubMedGoogle Scholar
  48. Motorin Y, Lyko F, Helm M. 5-methylcytosine in RNA: detection, enzymatic formation and biological functions. Nucleic Acids Res. 2010;38(5):1415–30. doi: 10.1093/nar/gkp1117.CrossRefPubMedGoogle Scholar
  49. Muller UF. Re-creating an RNA world. Cell Mol Life Sci. 2006;63(11):1278–93. doi: 10.1007/s00018-006-6047-1.CrossRefPubMedGoogle Scholar
  50. Nordlund ME, Johansson JO, von Pawel-Rammingen U, Bystrom AS. Identification of the TRM2 gene encoding the tRNA(m5U54)methyltransferase of Saccharomyces cerevisiae. RNA. 2000;6(6):844–60.CrossRefPubMedPubMedCentralGoogle Scholar
  51. Ny T, Bjork GR. Cloning and restriction mapping of the trmA gene coding for transfer ribonucleic acid (5-methyluridine)-methyltransferase in Escherichia coli K-12. J Bacteriol. 1980;142(2):371–9.PubMedPubMedCentralGoogle Scholar
  52. Pavlopoulou A, Kossida S. Phylogenetic analysis of the eukaryotic RNA (cytosine-5)-methyltransferases. Genomics. 2009;93(4):350–7. doi: 10.1016/j.ygeno.2008.12.004.CrossRefPubMedGoogle Scholar
  53. Pfaffeneder T, Hackner B, Truss M, Munzel M, Muller M, Deiml CA, et al. The discovery of 5-formylcytosine in embryonic stem cell DNA. Angewandte Chemie. 2011;50(31):7008–12. doi: 10.1002/anie.201103899.CrossRefPubMedGoogle Scholar
  54. Pfaffeneder T, Spada F, Wagner M, Brandmayr C, Laube SK, Eisen D, et al. Tet oxidizes thymine to 5-hydroxymethyluracil in mouse embryonic stem cell DNA. Nat Chem Biol. 2014;10(7):574–81. doi: 10.1038/nchembio.1532.CrossRefPubMedGoogle Scholar
  55. Racz I, Kiraly I, Lasztily D. Effect of light on the nucleotide composition of rRNA of wheat seedlings. Planta. 1978;142(3):263–7. doi: 10.1007/BF00385075.CrossRefPubMedGoogle Scholar
  56. Roberts RJ, Vincze T, Posfai J, Macelis D. REBASE--restriction enzymes and DNA methyltransferases. Nucleic Acids Res. 2005;33(Database issue):D230–2. doi: 10.1093/nar/gki029.CrossRefPubMedGoogle Scholar
  57. Santi DV. The mechanism and structure of thymidylate synthetase. Nucleic Acids Symp Ser. 1986;17:125–6.Google Scholar
  58. Santi DV, Hardy LW. Catalytic mechanism and inhibition of tRNA (uracil-5-)methyltransferase: evidence for covalent catalysis. Biochemistry. 1987;26(26):8599–606.CrossRefPubMedGoogle Scholar
  59. Schaefer M, Pollex T, Hanna K, Lyko F. RNA cytosine methylation analysis by bisulfite sequencing. Nucleic Acids Res. 2009;37(2):e12. doi: 10.1093/nar/gkn954.CrossRefPubMedGoogle Scholar
  60. Schiesser S, Hackner B, Pfaffeneder T, Muller M, Hagemeier C, Truss M, et al. Mechanism and stem-cell activity of 5-carboxycytosine decarboxylation determined by isotope tracing. Angewandte Chemie. 2012;51(26):6516–20. doi: 10.1002/anie.201202583.CrossRefPubMedGoogle Scholar
  61. Schiesser S, Pfaffeneder T, Sadeghian K, Hackner B, Steigenberger B, Schroder AS, et al. Deamination, oxidation, and C-C bond cleavage reactivity of 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine. J Am Chem Soc. 2013;135(39):14593–9. doi: 10.1021/ja403229y.CrossRefPubMedGoogle Scholar
  62. Selvadurai K, Wang P, Seimetz J, Huang RH. Archaeal Elp3 catalyzes tRNA wobble uridine modification at C5 via a radical mechanism. Nat Chem Biol. 2014;10(10):810–2. doi: 10.1038/nchembio.1610.CrossRefPubMedPubMedCentralGoogle Scholar
  63. Squires JE, Patel HR, Nousch M, Sibbritt T, Humphreys DT, Parker BJ, et al. Widespread occurrence of 5-methylcytosine in human coding and non-coding RNA. Nucleic Acids Res. 2012;40(11):5023–33. doi: 10.1093/nar/gks144.CrossRefPubMedPubMedCentralGoogle Scholar
  64. Tahiliani M, Koh KP, Shen Y, Pastor WA, Bandukwala H, Brudno Y, et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science. 2009;324(5929):930–5. doi: 10.1126/science.1170116.CrossRefPubMedPubMedCentralGoogle Scholar
  65. Urbonavicius J, Skouloubris S, Myllykallio H, Grosjean H. Identification of a novel gene encoding a flavin-dependent tRNA:m5U methyltransferase in bacteria--evolutionary implications. Nucleic Acids Res. 2005;33(13):3955–64. doi: 10.1093/nar/gki703.CrossRefPubMedPubMedCentralGoogle Scholar
  66. Vaught JD, Dewey T, Eaton BE. T7 RNA polymerase transcription with 5-position modified UTP derivatives. J Am Chem Soc. 2004;126(36):11231–7. doi: 10.1021/ja049009h.CrossRefPubMedGoogle Scholar
  67. Vaught JD, Bock C, Carter J, Fitzwater T, Otis M, Schneider D, et al. Expanding the chemistry of DNA for in vitro selection. J Am Chem Soc. 2010;132(12):4141–51. doi: 10.1021/ja908035g.CrossRefPubMedGoogle Scholar
  68. Wyatt GR, Cohen SS. The bases of the nucleic acids of some bacterial and animal viruses: the occurrence of 5-hydroxymethylcytosine. Biochem J. 1953;55(5):774–82.CrossRefPubMedPubMedCentralGoogle Scholar

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© Springer International Publishing Switzerland 2016

Authors and Affiliations

  1. 1.IMoPA UMR7365 CNRS-ULBioPole de l’Université de LorraineVandoeuvre-les-NancyFrance
  2. 2.Department of BiochemistryUniversity of Cape Coast, College of Agriculture and Natural Sciences, School of Biological SciencesCape CoastGhana
  3. 3.Institute of Pharmacy and BiochemistryJohannes Gutenberg University MainzMainzGermany

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