Circularly Permuted Variants Of Two Cg-Specific Prokaryotic Dna Methyltransferases

The highly similar prokaryotic DNA (cytosine-5) methyltransferases (C5-MTases) M.Mpel and M.Sssl share the specificity of eukaryotic C5-MTases (5'-CG), and can be useful research tools in the study of eukaryotic DNA methylation and epigenetic regulation. In an effort to improve the stability and solubility of complementing fragments of the two MTases, genes encoding circularly permuted (CP) variants of M.Mpel and M.Sssl were created, and cloned in a plasmid vector downstream of an arabinose-inducible promoter. MTase activity of the CP variants was tested by digestion of the plasmids with methylation-sensitive restriction enzymes. Eleven of the fourteen M.Mpel permutants and six of the seven M.Sssl permutants had detectable MTase activity as indicated by the full or partial protection of the plasmid carrying the cpMTase gene. Permutants cp62M.Mpel and cp58M.Sssl, in which the new N-termini are located between conserved motifs II and III, had by far the highest activity. The activity of cp62M.Mpel was comparable to the activity of wild-type M.Mpel. Based on the location of the split sites, the permutants possessing MTase activity can be classified in ten types. Although most permutation sites were designed to fall outside of conserved motifs, and the MTase activity of the permutants measured in cell extracts was in most cases substantially lower than that of the wild-type enzyme, the high proportion of circular permutation topologies compatible with MTase activity is remarkable, and is a new evidence for the structural plasticity of C5-MTases. A computer search of the REBASE database identified putative C5-MTases with CP arrangement. Interestingly, all natural circularly permuted C5-MTases appear to represent only one of the ten types of permutation topology created in this work.