On the substrate specificity of DNA methyltransferases -: Adenine-N6 DNA methyltransferases also modify cytosine residues at position N4

Methylation of DNA is important in many organisms and essential in mammals, Nucleobases can be methylated at the adenine-N-6, cytosine-N-4, or cytosine-C-5 atoms by specific DNA methyltransferases. We show here that the M.EcoRV, M.EcoRI, and Escherichia coli dam methyltransferases as well as the N- and C-terminal domains of the M.KokI enzyme, which were formerly all classified as adenine-N-6 DNA methyltransferases, also methylate cytosine residues at position N-4. Kinetic analyses demonstrate that the rate of methylation of cytosine residues by M.EcoRV and the M.FokI enzymes is reduced by only 1-2 orders of magnitude in relation to methylation of adenines, This result shows that although these enzymes methylate DNA in a sequence specific manner, they have a low substrate specificity with respect to the target base, This unexpected finding has implications on the mechanism of adenine-N-6 DNA methyltransferases, Sequence comparisons suggest that adenine-N-6 and cytosine-N-4 methyltransferases have changed their reaction specificity at least twice during evolution, a model that becomes much more likely given the partial functional overlap of both enzyme types. In contrast, methylation of adenine residues by the cytosine-N-4 methyltransferase M.BamHI was not detectable. On the basis of our results, we suggest that adenine-N-6 and cytosine-N-4 methyltransferases should be grouped into one enzyme family.