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 pyrimidin one have previously been shown to bind with a significantly greater affinit y to C5-cytosine DNA methyltransferases than unmodified DNA. Here, it is sh own that 2-H pyrimidinone, when incorporated into DNA duplexes containing t he recognition sites for M.HgaI-2 and M.MspI, elicits the formation of inhi bitory covalent nucleoprotein complexes. We have found that although covale nt complexes are formed between 2-H pyrimidinone-modified DNA and both M.Hg aI-2 and M.MspI, the kinetics of complex formation are quite distinct in ea ch case. Moreover, the formation of a covalent complex is still observed be tween 2-H pyrimidinone DNA and M.MspI in which the active-site cysteine res idue is replaced by serine or threonine. Covalent complex formation between M.MspI and 2-H pyrimidinone occurs as a direct result of nucleophilic atta ck by the residue at the catalytic position, which is enhanced by the absen ce of the 4-amino function in the base. The substitution of the catalytic c ysteine residue by tyrosine or chemical modification of the wildtype enzyme with N-ethylmaleimide, abolishes covalent interaction. Nevertheless the 2- H pyrimidinone-substituted duplex still binds to M.MspI with a greater affi nity than a standard cognate duplex, since the 2-H pyrimidinone base is mis -paired with guanine. (C) 1999 Academic Press.