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

P J Hurd, A J Whitmarsh, G S Baldwin, S M Kelly, J P Waltho, N C Price, B A Connolly, D P Hornby

    Research output: Contribution to journalArticlepeer-review

    Abstract

    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.

    Original languageEnglish
    Pages (from-to)389-401
    Number of pages13
    JournalJournal of molecular biology
    Volume286
    Issue number2
    DOIs
    Publication statusPublished - 19 Feb 1999

    Keywords

    • Binding Sites
    • Catalysis
    • Circular Dichroism
    • Cytidine
    • Cytosine
    • DNA (Cytosine-5-)-Methyltransferase
    • Enzyme Inhibitors
    • Magnetic Resonance Spectroscopy
    • Mutagenesis, Site-Directed
    • Protein Binding
    • Substrate Specificity

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