Mechanism-based inhibition of C5-cytosine DNA methyltransferases by 2-H pyrimidinone

J Mol Biol. 1999 Feb 19;286(2):389-401. doi: 10.1006/jmbi.1998.2491.


DNA duplexes in which the target cytosine base is replaced by 2-H pyrimidinone have previously been shown to bind with a significantly greater affinity to C5-cytosine DNA methyltransferases than unmodified DNA. Here, it is shown that 2-H pyrimidinone, when incorporated into DNA duplexes containing the recognition sites for M.HgaI-2 and M.MspI, elicits the formation of inhibitory covalent nucleoprotein complexes. We have found that although covalent complexes are formed between 2-H pyrimidinone-modified DNA and both M.HgaI-2 and M.MspI, the kinetics of complex formation are quite distinct in each case. Moreover, the formation of a covalent complex is still observed between 2-H pyrimidinone DNA and M.MspI in which the active-site cysteine residue is replaced by serine or threonine. Covalent complex formation between M.MspI and 2-H pyrimidinone occurs as a direct result of nucleophilic attack by the residue at the catalytic position, which is enhanced by the absence of the 4-amino function in the base. The substitution of the catalytic cysteine residue by tyrosine or chemical modification of the wild-type enzyme with N-ethylmaleimide, abolishes covalent interaction. Nevertheless the 2-H pyrimidinone-substituted duplex still binds to M.MspI with a greater affinity than a standard cognate duplex, since the 2-H pyrimidinone base is mis-paired with guanine.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Catalysis
  • Circular Dichroism
  • Cytidine / analogs & derivatives*
  • Cytidine / chemistry
  • Cytidine / pharmacology
  • Cytosine / chemistry
  • Cytosine / metabolism
  • DNA (Cytosine-5-)-Methyltransferases / antagonists & inhibitors*
  • Enzyme Inhibitors / pharmacology*
  • Magnetic Resonance Spectroscopy
  • Mutagenesis, Site-Directed
  • Protein Binding
  • Substrate Specificity


  • 1-ribofuranosyl-2(1H)-pyrimidinone
  • Enzyme Inhibitors
  • Cytidine
  • Cytosine
  • DNA (Cytosine-5-)-Methyltransferases