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-N6, cytosine-N4, or cytosine-C5 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. FokI enzyme, which were formerly all classified as adenine-N6 DNA methyltransferases, also methylate cytosine residues at position N4. 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-N6 DNA methyltransferases. 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.
Jeltsch
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A
Institut für Biochemie, Fachbereich Biologie, Heinrich-Buff-Ring 58, 35392 Giessen, Germany. Albert.Jeltsch@chemie.bio.uni-giessen.de
Christ
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F
Fatemi
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Roth
M
M
D000225:Adenine / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003596:Cytosine / Q000378:metabolism*; D015257:DNA-Cytosine Methylases / Q000378:metabolism; D004926:Escherichia coli; D019143:Evolution, Molecular; D007700:Kinetics; D008745:Methylation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism*; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D003596:Cytosine; C056127:DNA modification methylase EcoRV; C058668:DNA modification methylase FokI; D015257:DNA-Cytosine Methylases; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D000225:Adenine