Molecular Biology

, Volume 52, Issue 2, pp 272–278 | Cite as

Detection of DNA Methylation by Dnmt3a Methyltransferase using Methyl-Dependent Restriction Endonucleases

  • A. V. Sergeev
  • O. V. Kirsanova
  • A. G. Loiko
  • E. I. Nomerotskaya
  • E. S. Gromova
Molecular Cell Biology


DNA methylation at cytosine residues in CpG sites by DNA methyltransferases (MTases) is associated with various cell processes. Eukaryotic MTase Dnmt3a is the key enzyme that establishes the de novo methylation pattern. A new in vitro assay for DNA methylation by murine MTase Dnmt3a was developed using methyl-dependent restriction endonucleases (MD-REs), which specifically cleave methylated DNA. The Dnmt3a catalytic domain (Dnmt3a-CD) was used together with KroI and PcsI MD-REs. The assay consists in consecutive methylation and cleavage of fluorescently labeled DNA substrates, then the reaction products are visualized in polyacrylamide gel to determine the DNA methylation efficiency. Each MD-RE was tested with various substrates, including partly methylated ones. PcsI was identified as an optimal MDRE. PcsI recognizes two methylated CpG sites located 7 bp apart, the distance roughly corresponding to the distance between the active centers of the Dnmt3a-CD tetramer. An optimal substrate was designed to contain two methylated cytosine residues and two target cytosines in the orientation suitable for methylation by Dnmt3a-CD. The assay is reliable, simple, and inexpensive and, unlike conventional methods, does not require radioactive compounds. The assay may be used to assess the effectiveness of Dnmt3a inhibitors as potential therapeutic agents and to investigate the features of the Dnmt3a-CD function.


DNA methylation methyl-dependent restriction endonucleases eukaryotic DNA methyltransferases methylation efficiency 



C5-cytosine DNA methyltransferase


restriction endonuclease


methyl-dependent restriction endonuclease




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  1. 1.
    Bestor T.H. 2000. The DNA methyltransferases of mammals. Hum. Mol. Genet. 9, 2395–2402.CrossRefPubMedGoogle Scholar
  2. 2.
    Moore L.D., Le T., Fan G. 2013. DNA methylation and its basic function. Neuropsychopharmacology. 38, 23–38.CrossRefPubMedGoogle Scholar
  3. 3.
    Jurkowska R.Z., Jurkowski T.P., Jeltsch A. 2011. Structure and function of mammalian DNA methyltransferases. ChemBioChem. 12, 206–222.CrossRefPubMedGoogle Scholar
  4. 4.
    Bird A. 1999. DNA methylation de novo. Science. 286, 2287–2288.CrossRefPubMedGoogle Scholar
  5. 5.
    Gros C., Fahy J., Halby L., et al. 2012. DNA methylation inhibitors in cancer: Recent and future approaches. Biochimie. 94, 2280–2296.CrossRefPubMedGoogle Scholar
  6. 6.
    Jia D., Jurkowska R.Z., Zhang X., et al. 2007. Structure of Dnmt3a bound to Dnmt3L suggests a model for de novo DNA methylation. Nature. 449, 248–251.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Lukashevich O.V., Baskunov V.B., Darii M.V., et al. 2011. Dnmt3a-CD is less susceptible to bulky benzo[a]pyrene diol epoxide-derived DNA lesions than prokaryotic DNA methyltransferases. Biochemistry. 50, 875–881.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Yang L., Rau R., Goodell M.A. 2015. DNMT3A in haematological malignancies. Nat. Rev. Cancer. 15, 152–165.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Brennan C.A., Van Cleve M.D., Gumport R.I. 1986. The effects of base analogue substitutions on the methylation by the EcoRI modification methylase of octadeoxyribonucleotides containing modified EcoRI recognition sequences. J. Biol. Chem. 261, 7279–7286.PubMedGoogle Scholar
  10. 10.
    Roth M., Jeltsch A. 2000. Biotin–avidin microplate assay for the quantitative analysis of enzymatic methylation of DNA by DNA methyltransferases. Biol. Chem. 381, 269–272.CrossRefPubMedGoogle Scholar
  11. 11.
    Hübscher U., Pedrali-Noy G., Knust-Kron B., et al. 1985. DNA methyltransferases: Activity minigel analysis and determination with DNA covalently bound to a solid matrix. Anal. Biochem. 150, 442–448.CrossRefPubMedGoogle Scholar
  12. 12.
    Jeltsch A., Friedrich T., Roth M. 1998. Kinetics of methylation and binding of DNA by the EcoRV adenine-N6 methyltransferase. J. Mol. Biol. 275, 747–758.CrossRefPubMedGoogle Scholar
  13. 13.
    Jurkowska R.Z., Ceccaldi A., Zhang Y., et al. 2011. DNA methyltransferase assays. Epigenet. Protoc. 791, 157–177.CrossRefGoogle Scholar
  14. 14.
    Ivanov A.A., Koval V.S., Susova O.Y., et al. 2015. DNA specific fluorescent symmetric dimeric bisbenzimidazoles DBP(n): The synthesis, spectral properties, and biological activity. Bioorg. Med. Chem. Lett. 25, 2634–2638.CrossRefPubMedGoogle Scholar
  15. 15.
    Cherepanova N.A., Ivanov A.A., Maltseva D.V., et al. 2011. Dimeric bisbenzimidazoles inhibit the DNA methylation. catalyzed by the murine Dnmt3a catalytic domain. J. Enzym. Inhib. Med. Chem. 26, 295–300.Google Scholar
  16. 16.
    Zemlyanskaya E.V., Degtyarev S.K. 2013. Substrate specificity and properties of methyl-directed site-specific DNA endonucleases. Mol. Biol. (Moscow). 47 (6), 784–795.CrossRefGoogle Scholar
  17. 17.
    Chernukhin V.A., Kileva E.V., Tomilova Yu.E., et al. 2011. New methyl-dependent site-specific endonuclease KroI recognizes and cleaves 5'-G C (5mC)GGC-3' DNA sequence. Vestn. Biotekhnol. Fiz.-Khim. Biol. im. Yu.A. Ovchinnikova. 7, 14–20.Google Scholar
  18. 18.
    Chernukhin V.A., Nayakshina T.N., Tarasova G.V., et al. 2009. RF Patent 2377294.Google Scholar
  19. 19.
    Baskunov V.B., Subach F.V., Kolbanovskiy A., et al. 2005. Effects of benzo[a]pyrene-deoxyguanosine lesions on DNA methylation catalyzed by EcoRII DNA methyltransferase and on DNA cleavage effected by EcoRII restriction endonuclease. Biochemistry. 44, 1054–1066.CrossRefPubMedGoogle Scholar
  20. 20.
    Darii M.V., Cherepanova N.A., Subach O.M., et al. 2009. Mutational analysis of the CG recognizing DNA methyltransferase SssI: Insight into enzyme–DNA interactions. Biochim. Biophys. Acta, Proteins Proteomics. 1794, 1654–1662.CrossRefGoogle Scholar
  21. 21.
    Jurkowska R.Z., Siddique A.N., Jurkowski T.P., Jeltsch A. 2011. Approaches to enzyme and substrate design of the murine Dnmt3a DNA methyltransferase. ChemBioChem. 12, 1589–1594.CrossRefPubMedGoogle Scholar
  22. 22.
    Kirsanova O.V., Sergeev A.V., Yasko I.S., Gromova E.S. 2017. The impact of 6-thioguanine incorporation into DNA on the function of DNA methyltransferase Dnmt3a. Nucleosides, Nucleotides Nucleic Acids 36, 392–405.CrossRefPubMedGoogle Scholar
  23. 23.
    Cohen-Karni D., Xu D., Apone L., et al. 2011. The MspJI family of modification-dependent restriction endonucleases for epigenetic studies. Proc. Natl. Acad. Sci. U. S. A. 108, 11040–11045.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Oakes C.C., La Salle S., Robaire B., Trasler J.M. 2006. Evaluation of a quantitative DNA methylation analysis technique using methylation-sensitive/dependent restriction enzymes and real-time PCR. Epigenetics. 1, 146–152.CrossRefPubMedGoogle Scholar
  25. 25.
    Yokochi T., Robertson K.D. 2002. Preferential methylation of unmethylated DNA by mammalian de novo DNA methyltransferase Dnmt3a. J. Biol. Chem. 277, 11735–11745.CrossRefPubMedGoogle Scholar

Copyright information

© Pleiades Publishing, Inc. 2018

Authors and Affiliations

  • A. V. Sergeev
    • 1
  • O. V. Kirsanova
    • 1
  • A. G. Loiko
    • 1
  • E. I. Nomerotskaya
    • 1
  • E. S. Gromova
    • 1
  1. 1.Chemical FacultyMoscow State UniversityMoscowRussia

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