DETERMINATION OF THE ORDER OF SUBSTRATE ADDITION TO MSPI DNA METHYLTRANSFERASE USING A NOVEL MECHANISM-BASED INHIBITOR

The cloning and overexpression of the MspI DNA methyltransferase as a functional fusion with glutathione S-transferase is described. The fus ion enzyme retains full biological activity and has been used to inves tigate the interaction of substrates and inhibitors with MspI DNA meth yltransferase. The fusion enzyme has been purified to homogeneity in a single step on GSH-agarose and is free from contaminating exonuclease activity. The enzyme can be photolabelled with S-adenosyl-L-methionin e and the level of incorporation of label is enhanced by the presence of a nonspecific DNA duplex. In the presence of a cognate oligodeoxynu cleotide, no photolabelling was observed since methyl transfer occurs instead. The inclusion of a mechanism-based inhibitor of C-5 deoxycyti dine DNA methylation (an oligodeoxynucleotide containing the base 2-py rimidinone-1-beta-D-2'-deoxyribofuranoside in the position of the deox ycytidine to which methyl addition occurs), which is thought to form a covalent interaction with the reactive cysteine of such enzymes, led to an enhancement of S-adenosyl-L-methionine photolabelling which sugg ests that, in contrast with results obtained with EcoRII DNA methyltra nsferase [Som and Friedman (199 1) J. Biol. Chem. 266, 2937-2945), met hylcysteine is not the photolabelled product. The implications of the results obtained with this mechanism-based inhibitor are discussed wit h respect to other C-5-specific DNA methyltransferases. Gel-retardatio n assays in the presence of cognate oligodeoxynucleotides that contain the reactive pyrimidinone base in place of the deoxycytidine target b ase are described. These demonstrate that most probably a stable coval ent bond is formed between the methyltransferase and this oligodeoxynu cleotide. However, the alternative of extremely tight non-covalent bin ding cannot be rigorously excluded. Furthermore, the results from thes e experiments indicate that the reaction mechanism proceeds in a manne r similar to that of HhaI DNA methyltransferase with sequence-specific DNA binding being followed by addition of S-adenosyl-L-methionine and concomitant isomerization of the ternary complex leading to methyl tr ansfer. S-Adenosyl-L-homocysteine appears to inhibit the reaction path way as a result of either competition with the methyl donor and potent iation of a high-affinity interaction between the enzyme and DNA in an abortive ternary complex or through an allosteric interaction.