Mechanism-based inhibition of C5-cytosine DNA methyltransferases by 2-H pyrimidinone.
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.
Hurd
P J
PJ
Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Sheffield, Western Bank, S10 2TN, UK.
Whitmarsh
A J
AJ
Baldwin
G S
GS
Kelly
S M
SM
Waltho
J P
JP
Price
N C
NC
Connolly
B A
BA
Hornby
D P
DP
D001665:Binding Sites; D002384:Catalysis; D002942:Circular Dichroism; D003562:Cytidine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D003596:Cytosine / Q000737:chemistry / Q000378:metabolism; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000037:antagonists & inhibitors*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D013379:Substrate Specificity