Cytosine-to-Uracil Deamination by SssI DNA Methyltransferase

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 Type II restriction endonuclease MvaI has dual specificity

The MvaI restriction endonuclease cuts 5′-CC↓AGG-3′/5′-CC↑TGG-3′ sites as indicated by the arrows. N4-methylation of the inner cytosines (Cm4CAGG/Cm4CTGG) protects the site against MvaI cleavage. Here, we show that MvaI nicks the G-strand of the related sequence (CCGGG/CCCGG, BcnI site) if the inner cytosines are C5-methylated: Cm5C↓GGG/CCm5CGG. At M.SssI-methylated SmaI sites, where two oppositely oriented methylated BcnI sites partially overlap, double-nicking leads to double-strand cleavage (CCm5C↓GGG/CCm5C↑GGG) generating fragments with blunt ends. The double-strand cleavage rate and the stringency of substrate site recognition is lower at the methylation-dependent site than at the canonical target site. MvaI is the first restriction endonuclease shown to possess, besides the ‘normal’ activity on its unmethylated recognition site, also a methylation-directed activity on a different sequence

Cytosine deamination by M.SssI <i>in</i><i>vitro</i>.

<p>pUP41 (Ap^R, Kn^S) plasmid DNA was incubated with or without wild-type M.SssI (2-fold molar excess relative to CG sites) at 30°C for 4 h, and frequency of C-to-U deamination was determined by scoring the numbers of Kn^R and Ap^R transformants in <i>E. coli</i> ER2357 <i>ung</i> strain. SAM (160 µM) and 5’-amino-5’-deoxyadenosine (AA, 250 μM) were added to samples as indicated. Error bars represent standard error of the mean of at least three independent experiments (p<0.01).</p

Estimation of DNA MTase activity of the F17S and G19D M.SssI mutants by restriction enzyme protection assay.

<div><p>Lambda phage DNA was incubated with different concentrations of WT and mutant M.SssI in the presence of SAM as described in Materials and Methods. Methylation status of the DNA was subsequently tested by digestion with the methylation sensitive restriction enzyme Hin6I, and the digestion was analyzed by agarose gel electrophoresis. Lane Undig., undigested plasmid; M, molecular weight marker (GeneRuler 1 kb Plus and GeneRuler 1 kb DNA Ladders, Fermentas).</p> <p>M.SssI-mediated cytosine deamination <i>in </i><i>vivo</i> was initially investigated using a two-plasmid-system, with the <i>E. coli</i> host containing the indicator plasmid pUP41 and one of the M.SssI-expressing plasmids pSTdC-MSssI, pSTdC-MSssI(F17S) or pSTdC-MSssI(G19D). The latter plasmids have pSC101 replicon and are compatible with pUP41. We observed elevated reversion frequency to kanamycin resistance with the Ung^- host ER2357 expressing the G19D variant (not shown). </p></div

DNA methyltransferase activity of the F17S and G19D M.SssI mutants.

<div><p>(<b>A</b>) Amino acid sequence alignment between segments of M.HpaII and M.SssI. Conserved residues of the FXGXG motif are in bold. The F17S and G19D substitutions are indicated below the sequence. </p> <p>(<b>B</b>) Hin6I digestion of plasmids pBHNS-MSssI, pBHNS-MSssI(F17S) and pBHNS-MSssI(G19D) encoding WT or mutant M.SssI variants, respectively as indicated above the lanes. Plasmids were isolated from cultures grown for 4-6-8 h in the presence of arabinose to induce M.SssI expression. Resistance to Hin6I (recognition sequence GCGC) indicates M.SssI-specific methylation (R. Kazlauskiene, cited in REBASE [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079003#B45" target="_blank">45</a>]. Lane Unmeth., unmethylated pBHNS-MSssI(G19D) isolated from cells grown in the presence of glucose; Lane Undig., undigested pBHNS-MSssI; M, molecular weight marker (GeneRuler 1 kb DNA Ladder, Fermentas). </p> <p>(<b>C</b>) Effect of WT and mutant M.SssI production on growth of <i>E. coli </i><i>mcrBC</i> and <i>mcrBC</i>⁺ hosts. DH10B <i>mcrBC</i> contained pBHNS-MSssI, pBHNS-MSssI(F17S) or pBHNS-MSssI(G19D) as indicated. DH5α <i>mcrBC</i>^<i>+</i> contained pBHNS-MSssI(F17S) or pBHNS-MSssI(G19D). Bacteria were grown in LB/Ap medium at 30°C. MTase expression was induced at time 0 by arabinose. Error bars represent standard error of the mean of three independent experiments. </p></div

Effect of sinefungin and 5’-amino-5’-deoxyadenosine on cytosine deamination activity of M.SssI and its mutants.

<p>Plasmid pUP41 was incubated with purified M.SssI, M.SssI(F17S) or M.SssI(G19D) and frequency of Kn^R revertants was determined in ER2357 <i>ung</i> strain as described in Materials and Methods and legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079003#pone-0079003-g001" target="_blank">Figure 1</a>. Sinefungin (SF) was used at 500 and 5’-amino-5’-deoxyadenosine (AA) at 250 µM concentration in samples indicated below the bars. Reversion frequency was derived from the ratio of the Kn^R and Ap^R transformants. Striped bar, no enzyme; empty bars, wild-type M.SssI; grey bars, M.SssI(F17S); black bars, M.SssI(G19D). Error bars represent standard error of the mean of five independent experiments (p<0.01).</p