Human Genetics

, Volume 83, Issue 2, pp 181–188

Cytosine methylation and the fate of CpG dinucleotides in vertebrate genomes

  • David N. Cooper
  • Michael Krawczak
Original Investigations


The dinucleotide CpG is a “hotspot” for mutation in the human genome as a result of (1) the modification of the 5′ cytosine by cellular DNA methyltransferases and (2) the consequent high frequency of spontaneous deamination of 5-methyl cytosine (5mC) to thymidine. DNA methylation thus contributes significantly, albeit indirectly, to the incidence of human genetic disease. We have attempted to estimate for the first time the in vivo rate of deamination of 5mC from the measured rate of 5mC deamination in vitro and the known error frequency of the cellular G/T mismatch-repair system. The accuracy and utility of this estimate (md) was then assessed by comparison with clinical data, and an improved estimate of md (1.66x10-16 s-1) was derived. Comparison of the CpG mutation rates exibited by globin gene and pseudogene sequences from human, chimpanzee and macaque provided further estimates of md, all of which were consistent with the first. Use of this value in a mathematical model then permitted the estimation of the length of time required to produce the level of “CpG suppression” currently found in the “bulk DNA” of vertebrate genomes. This time span, approximately 450 million years, corresponds closely to the estimated time since the emergence and adaptive radiation of the vertebrates and thus coincides with the probable advent of heavily methylated genomes. An accurate estimate of the 5mC deamination rate is important not only for clinical medicine but also for studies of gene evolution. Our data suggest both that patterns of vertebrate gene methylation may be comparatively stable over relatively long periods of evolutionary time, and that the rate of CpG deamination can, under certain limited conditions, serve as a “molecular clock”.


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  1. Adams RLP, Eason R (1984) Increased CpG content of DNA stabilizes CpG dinucleotides. Nucleic Acids Res 12:5867–5877Google Scholar
  2. Adams RLP, Davis T, Rinaldi A, Eason R (1987) CpG deficiency, dinucleotide distributions and nucleosome positioning. Eur J Biochem 165:107–115Google Scholar
  3. Bird AP (1980) DNA methylation and the frequency of CpG in animal DNA. Nucleic Acids Res 8:1499–1504Google Scholar
  4. Bird AP (1986) CpG-rich islands and the function of DNA methylation. Nature 321:209–213Google Scholar
  5. Bird AP (1987) CpG islands as gene markers in the vertebrate nucleus. Trends Genet 3:342–347Google Scholar
  6. Bird AP, Taggart MH, Nichols RD, Higgs DR (1987) Non-methylated CpG-rich islands at the human α-globin locus: implications for evolution of the α-globin pseudogene. EMBO J 6:999–1004Google Scholar
  7. Bolden AH, Nalin CM, Ward CA, Poonian MS, Weissbach A (1986) Primary DNA sequence determines sites of maintenance and de novo methylation by mammalian DNA methyltransferase. Mol Cell Biol 6:1135–1140Google Scholar
  8. Britten RJ (1986) Rates of DNA sequence evolution differ between taxonomic groups. Science 235:1393–1398Google Scholar
  9. Brown TC, Jiricny J (1987) A specific mismatch repair event protects mammalian cells from loss of 5-methylcytosine. Cell 50:945–950Google Scholar
  10. Brown TC, Jiricny J (1988) Different base/base mispairs are corrected with different efficiencies and specificites in monkey kidney cells. Cell 54:705–711Google Scholar
  11. Brown WRA, Bird AP (1986) Long-range restriction site mapping of mammalian genomic DNA. Nature 322:477–481Google Scholar
  12. Colbert EH (1969) Evolution of the vertebrates. Wiley, New YorkGoogle Scholar
  13. Cooper DN (1983) Eukaryotic DNA methylation. Hum Genet 64:315–333Google Scholar
  14. Cooper DN, Gerber-Huber S (1985) DNA methylation and CpG suppression. Cell Differ 17:199–205Google Scholar
  15. Cooper DN, Youssoufian H (1988) The CpG dinucleotide and human genetic disease. Hum Genet 78:151–155Google Scholar
  16. Cooper DN, Taggart MH, Bird AP (1983) Unmethylated domains in vertebrate DNA. Nucleic Acids Res 11: 647–658Google Scholar
  17. Cooper DN, Gerber-Huber S, Nardelli D, Schubiger JL, Wahli W (1987) The distribution of the dinucleotide CpG and cytosine methylation in the vitellogenin gene family. J Mol Evol 25:107–115Google Scholar
  18. Coulondre C, Miller JH, Farabaugh PJ, Gilbert W (1978) Molecular basis of base substitution hotspots in Escherichia coli. Nature 274:775–780Google Scholar
  19. Crow FJ, Denniston C (1985) Mutation in human populations. Adv Hum Genet 14:59–123Google Scholar
  20. Duncan BK, Miller JH (1980) Mutagenic deamination of cytosine residues in DNA. Nature 287:560–561Google Scholar
  21. Easteal S (1988) Rate constancy of globin gene evolution in placental mammals. Proc Natl Acad Sci USA 85:7622–7626Google Scholar
  22. Ehrlich M, Norris KF, Wang RYH, Kuo KC, Gehrke CW (1986) DNA cytosine methylation and heat-induced deamination. Biosci Rep 6:387–393Google Scholar
  23. Estivill X, Farrall M, Scambler PJ, Bell GM, Hawley KMF, Lench NJ, Bates GP, Kruyer HC, Frederick PA, Stanier P, Watson EK, Williamson R, Wainwright BJ (1987) A candidate for the cystic fibrosis locus isolated by selection for methylation-free islands. Nature 326:840–845Google Scholar
  24. Gardiner-Garden M, Frommer M (1987) CpG islands in vertebrate genomes. J Mol Biol 196:261–282Google Scholar
  25. Grippo P, Iaccarino M, Parisi E, Scarano E (1968) Methylation of DNA in developing sea urchin embryos. J Mol Biol 36:195–208Google Scholar
  26. Harbers K, Harbers B, Spencer JH (1975) Nucleotide clusters in deoxyribonucleic acids. XII. The distribution of 5-methylcytosine in pyrimidine oligonucleotides of mouse l-cell satellite DNA and main band DNA. Biochem Biophys Res Commun 66:738–746Google Scholar
  27. Hare JT, Taylor JH (1985) One role for DNA methylation in vertebrate cells is strand discrimination in mismatch repair. Proc Natl Acad Sci USA 82:7350–7354Google Scholar
  28. Jones M, Wagner R, Radman M (1987) Mismatch repair of deaminated 5-methyl-cytosine. J Mol Biol 194:155–159Google Scholar
  29. Josse J, Kaiser AD, Kornberg A (1961) Enzymatic synthesis of deoxyribonucleic acid. VIII. Frequencies of nearest neighbour base sequences in deoxyribonucleic acid. J Biol Chem 236:864–875Google Scholar
  30. Kolsto AB, Kallias G, Giguere V, Isobe KI, Prydz H, Grosveld F (1986) The maintenance of methylation-free islands in transgenic mice. Nucleic Acids Res 14:9667–9677Google Scholar
  31. Kramer B, Kramer W, Fritz HJ (1984) Different base/base mismatches are corrected with different efficiencies by the methyl-directed DNA mismatch repair system of E. coli. Cell 38:879–887Google Scholar
  32. Kunkel TA, Alexander PS (1986) The base substitution fidelity of eukaryotic DNA polymerase. J Biol Chem 261:160–166Google Scholar
  33. Li WH, Tanimura M (1987) The molecular clock runs more slowly in man than in apes and monkeys. Nature 326:93–96Google Scholar
  34. Lindahl T (1982) DNA repair enzymes. Annu Rev Biochem 51:61–87Google Scholar
  35. Lindahl T, Nyberg B (1974) Heat-induced deamination of cytosine residues in deoxyribonucleic acid. Biochemistry 13:3405–3410Google Scholar
  36. Lindsay S, Bird AP (1987) Use of restriction enzymes to select potential gene sequences in mammalian DNA Nature 327:336–338Google Scholar
  37. Lu AL, Welsh K, Clark S, Su SS, Modrick P(1984) Repair of DNA base-pair mismatches in extracts of Escherichia coli. Cold Spring Harbor Symp Quant Biol 49:589–596Google Scholar
  38. Mavilio F, Giampaolo A, Caré A, Migliaccio G, Calandrini M, Russo G, Pagliardi GL, Mastroberardino G, Marinucci M, Peschle C (1983) Molecular mechanisms of human hemoglobin switching; selective undermethylation and expression of globin genes in embryonic, fetal and adult erythroblasts. Proc Natl Acad Sci USA 80:6907–6911Google Scholar
  39. Mazin AL, Vanyushin BF (1987) Loss of CpG dinucleotides from DNA. III. Methylation and evolution of histone genes. Mol Biol (Mosk) 21:566–574Google Scholar
  40. Monk M (1988) Genomic imprinting. Genes Dev 2:921–925Google Scholar
  41. Miyamoto MM, Koop BF, Slightom JL, Goodman M, Tennant MR (1988) Molecular systematics of higher primates: genealogical relations and classification. Proc Natl Acad Sci USA 85:7627–7631Google Scholar
  42. Nussinov R (1981) Eukaryotic dinucleotide preference rules and their implications for degenerate codon usage. J Mol Biol 149:125–131Google Scholar
  43. Pech M, Streck RE, Zachau HG (1979) Patchwork structure of a bovine satellite DNA. Cell 18:883–893Google Scholar
  44. Ploeg LHT van der, Flavell RA (1980) DNA methylation in the human γδβ-globin locus in erythroid and non-erythroid tissues. Cell 19:947–958Google Scholar
  45. Poustka A, Pohl T, Barlow DP, Zehetner G, Craig A, Michiels F, Ehrich E, Frischauf AM, Lehrach H (1986) Molecular approaches to mammalian genetics. Cold Spring Harbor Symp Quant Biol 51:131–139Google Scholar
  46. Razin A, Szyf M, Kafri T, Roll M, Giloh H, Scarpa S, Carotti D, Cantoni GL (1986) Replacement of 5-methylcytosine by cytosine: a possible mechanism for transient DNA demethylation during differentiation. Proc Natl Acad Sci USA 83:2827–2831Google Scholar
  47. Salomon R, Kaye AM (1970) Methylation of mouse DNA in vivo: DI and tripyrimidine sequences containing 5-methylcytosine. Biochim Biophys Acta 204:340–351Google Scholar
  48. Savatier P, Trabuchet G, Fauré C, Cheblouré Y, Gouy M, Verdier G, Nigon VM (1985) Evolution of the primate beta-globin gene region. High rate of variation in CpG dinucleotides and in short repeated sequences between man and chimpanzee. J Mol Biol 182:21–29Google Scholar
  49. Selker EV, Stevens JN (1985) DNA methylation at asymetric sites is associated with numerous transition mutations. Proc Natl Acad Sci USA 82:8114–8118Google Scholar
  50. Setlow P (1976) Nearest neighbour frequencies in deoxyribonucleic acids. In: Fasman GD (ed) CRC handbook of biochemistry and molecular biology, vol 2: Nucleic acids, 3rd edn. CRC Press, Cleveland, OhioGoogle Scholar
  51. Shenoy S, Ehrlich KC, Ehrlich M (1987) Repair of thymidine-guanine and uracil-guanine mismatched base-pairs in bacteriophage M13 mp18 DNA heteroduplexes. J Mol Biol 197:617–626Google Scholar
  52. Shmookler-Reis RJ, Goldstein S (1982) Variability of DNA methylation patterns during serial passage of human diploid fibroblasts. Proc Natl Acad Sci USA 79:3949–3953Google Scholar
  53. Sibley CG, Ahlquist JE (1984) The phylogeny of the hominoid primates as indicated by DNA-DNA hybridization. J Mol Evol 20:2–15Google Scholar
  54. Taylor JH (1984) DNA methylation and cellular differentiation. Springer, Wien New YorkGoogle Scholar
  55. Vogel F, Kopun M (1977) Higher frequencies of transitions among point mutations. J Mol Evol 9:159–180Google Scholar
  56. Vogel F, Motulsky AG (1986) Human genetics — problems and approaches, 2nd edn. Springer, Berlin Heidelberg New YorkGoogle Scholar
  57. Vogel F, Röhrborn G (1965) Mutationsvorgänge bei der Entstehung von Hämoglobinvarianten. Humangenetik 1:635–650Google Scholar
  58. Vogel F, Kopun M, Rathenburg R (1976) Mutation and molecular evolution. In: Goodman M, Tashian RE, Tashian JH (eds) Molecular anthropology. Plenum Press, New YorkGoogle Scholar
  59. Wang RYH, Kuo KC, Gehrke CW, Huang LH, Ehrlich M (1982) Heat and alkali-induced deamination of 5-methylcytosine and cytosine residues in DNA. Biochim Biophys Acta 697:371–377Google Scholar
  60. Weber E (1980) Grundriß der biologischen Statistik, 8th edn. Fischer, StuttgartGoogle Scholar
  61. Woodcock DM, Crowther PJ, Diver WP (1987) The majority of methylated deoxycytidines in human DNA are not in the CpG dinucleotide. Biochem Biophys Res Commun 145:888–894Google Scholar
  62. Young JZ (1973) The life of mammals, 2nd edn. Clarendon Press, OxfordGoogle Scholar
  63. Zell R, Fritz JH (1987) DNA mismatch-repair in Escherichia coli counteracting the hydrolytic deamination of 5-methylcytosine residues. EMBO J 6:1809–1815Google Scholar

Copyright information

© Springer-Verlag 1989

Authors and Affiliations

  • David N. Cooper
    • 1
  • Michael Krawczak
    • 2
  1. 1.Thrombosis Research Unit, King's College School of Medicine and DentistryUniversity of LondonLondonUK
  2. 2.Institut für Humangenetik der UniversitätGöttingenGermany

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