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 language | English |
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Pages (from-to) | 389-401 |
Number of pages | 13 |
Journal | Journal of molecular biology |
Volume | 286 |
Issue number | 2 |
DOIs | |
Publication status | Published - 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