Multicopy clones of Escherichia coli cytosine methyltransferases Dcm and EcoRII methylase (M. EcoRII) cause 50-fold increase in C T mutations at their canonical site of methylation, 5`-CmeCAGG (meC is 5-methylcytosine). These plasmids also cause transition mutations at the second cytosine in the sequences CCGGG at 10-fold lower frequency. Similarly, M. HpaII was found to cause a significant increase in C T mutations at a CCAG site, in addition to causing mutations at its canonical site of methylation, CCGG. Using a plasmid that substantially overproduces M. EcoRII, in vivo methylation at CCSGG (S is C or G) and other non-canonical sites could be detected using a gel electrophoretic assay. There is a direct correlation between the level of M. EcoRII activity in cells, the extent of methylation at non-canonical sites and frequency of mutations at these same sites. Overproduction of M. EcoRII in cells also causes degradation of DNA and induction of the SOS response. In vitro, M. EcoRII methylates an oligonucleotide duplex containing a CCGGG site at a slow rate, suggesting that overproduction of the enzyme is essential for significant amounts of such methylation to occur. Together these results show that cytosine methyltransferases occasionally methylate cellular DNA at non-canonical sites and suggest that in E. coli, methylation-specific restriction systems and sequence specificity of the DNA mismatch correction systems may have evolved to accommodate this fact. These results also suggest that mutational effects of cytosine methyltransferases may be much broader than previously imagined.

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