On the Substrate Specificity of DNA Methyltransferases

Abstract

Methylation of DNA is important in many organisms and essential in mammals. Nucleobases can be methyl- ated at the adenine-N6, cytosine-N4, or cytosine-C5 atoms by specific DNA methyltransferases. We show here that the M.EcoRV, M.EcoRI, and Escherichia coli dam meth- yltransferases as well as the N- and C-terminal domains of the M.FokI enzyme, which were formerly all classified as adenine-N6 DNA methyltransferases, also methylate cytosine residues at position N4. Kinetic analyses dem- onstrate that the rate of methylation of cytosine resi- dues 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 en- zymes 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-N6 DNA methyltrans- ferases. Sequence comparisons suggest that adenine-N6 and cytosine-N4 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-N4 methyltransferase M.BamHI was not detectable. On the basis of our results, we suggest that adenine-N6 and cytosine-N4 methyltransferases should be grouped into one enzyme family.