title: Cytosine-to-uracil deamination by SssI DNA methyltransferase.
abstract: The prokaryotic DNA(cytosine-5)methyltransferase M.SssI shares the specificity of eukaryotic DNA methyltransferases (CG) and is an important model and experimental tool in the study of eukaryotic DNA methylation. Previously, M.SssI was shown to be able to catalyze deamination of the target cytosine to uracil if the methyl donor S-adenosyl-methionine (SAM) was missing from the reaction. To test whether this side-activity of the enzyme can be used to distinguish between unmethylated and C5-methylated cytosines in CG dinucleotides, we re-investigated, using a sensitive genetic reversion assay, the cytosine deaminase activity of M.SssI. Confirming previous results we showed that M.SssI can deaminate cytosine to uracil in a slow reaction in the absence of SAM and that the rate of this reaction can be increased by the SAM analogue 5'-amino-5'-deoxyadenosine. We could not detect M.SssI-catalyzed deamination of C5-methylcytosine ((m5)C). We found conditions where the rate of M.SssI mediated C-to-U deamination was at least 100-fold higher than the rate of (m5)C-to-T conversion. Although this difference in reactivities suggests that the enzyme could be used to identify C5-methylated cytosines in the epigenetically important CG dinucleotides, the rate of M.SssI mediated cytosine deamination is too low to become an enzymatic alternative to the bisulfite reaction. Amino acid replacements in the presumed SAM binding pocket of M.SssI (F17S and G19D) resulted in greatly reduced methyltransferase activity. The G19D variant showed cytosine deaminase activity in E. coli, at physiological SAM concentrations. Interestingly, the C-to-U deaminase activity was also detectable in an E. coli ung (+) host proficient in uracil excision repair.
authors.affiliation: Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary.
authors.forename: Ildikó
authors.initials: I
authors.lastname: Stier
authors.forename: Antal
authors.initials: A
authors.lastname: Kiss
mesh_terms: D001665:Binding Sites; D003596:Cytosine / Q000737:chemistry / Q000378:metabolism*; D015257:DNA-Cytosine Methylases / Q000502:physiology*; D003641:Deamination; D003839:Deoxyadenosines / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D008957:Models, Genetic; D012436:S-Adenosylmethionine / Q000737:chemistry / Q000378:metabolism; D014498:Uracil / Q000737:chemistry / Q000378:metabolism*
chemical_list: D003839:Deoxyadenosines; C057928:5'-amino-5'-deoxyadenosine; D014498:Uracil; D012436:S-Adenosylmethionine; D003596:Cytosine; C081573:DNA modification methylase SssI; D015257:DNA-Cytosine Methylases