Kinetic studies of MvaI DNA methyltransferase interaction with modified oligonucleotide duplexes

We have measured steady-state kinetics of a N4-cytosine methylase, M.MvaI, using as substrates modified non-selfcomplementary tetradecanucleotide duplexes containing the CCWGG target sequence. The inner or outer localisation of the dI residue in the MvaI recognition site seems to be of little importance since the specificity constants kcat/KM are only 2 to 7 fold smaller than that of the canonical substrate. Replacement of dG residues by dI in both strands resulted in a 25 to 60-fold decrease of the specificity constant. Modifications of the phosphate backbone or opening of the sugar ring of one of the dG residues had only little influence on the action of M.MvaI. The enzyme appears to be rather tolerant to different kinds of modifications in its substrate in the mainor groove.