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 (m5C). We found conditions where the rate of M.SssI mediated C-to-U deamination was at least 100-fold higher than the rate of m5C-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.

The Genomic Impact of DNA CpG Methylation on Gene Expression; Relationships in Prostate Cancer

“…Methylated cytosine is a dynamic modification not only because it is added and removed by enzymatic processes, but also because it can spontaneously deaminate and, therefore, mutate [15,16,17]. By contrast, spontaneous deamination of unmethylated cytosine is readily recognized by the DNA repair machinery and corrected.…”

DNA demethylation pathways: Additional players and regulators

“…At least one human APOBEC3 paralog (3A, but not 3G) indeed deaminates 5mC in DNA faster than AID [57], but the single murine APOBEC3 also discriminates against 5mC [52]. Some prokaryotic methyltransferases can catalyze deamination of Cs to uracils [58] and of 5mCs to thymines [59] in the absence of S-adenosylmethionine (SAM), but mammalian DNA methyltransferases are not known to catalyze this reaction, either in vitro or in vivo. Moreover, the implication of alternative deaminases does not explain the genetic data that implicate specifically Aid and not deaminases in general in DNA demethylation.…”

Simultaneous Methylation-Level Assessment of Hundreds of CpG Sites by Targeted Bisulfite PCR Sequencing (TBPseq)

“…The results from amplicon sequencing showed that the mean methylation level was 60.4 ± 9.9% when the Sss I-remethylated DNA template was used, whereas the levels were 1.6 ± 2.5% and 29.8 ± 8.2% when the demethylated DNA template only or an equal mixture of remethylated and demethylated DNA, respectively, was used ( Figure 1C ). The 60% methylation level obtained from the use of the remethylated DNA indicated an incomplete methylation, possibly by insufficient Sss I enzyme activity or Sss I-catalyzed cytosine-to-uracil conversion in SAM deficient condition ( Bandaru et al, 1995 ; Zingg et al, 1996 ; Stier and Kiss, 2013 ). Nevertheless, the 30% methylation level acquired from the 1:1 mixed DNA template implied that the methylation frequencies of target CpGs at genomic regions were well-preserved even after the multiplex PCR and library preparation processes during TBPseq.…”