Circularly permuted variants of two CG-specific prokaryotic DNA methyltransferases

Abstract

The highly similar prokaryotic DNA (cytosine-5) methyltransferases (C5-MTases) M.MpeI and M.SssI 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.MpeI and M.SssI 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.MpeI permutants and six of the seven M.SssI permutants had detectable MTase activity as indicated by the full or partial protection of the plasmid carrying the cpMTase gene. Permutants cp62M.MpeI and cp58M.SssI, in which the new N-termini are located between conserved motifs II and III, had by far the highest activity. The activity of cp62M.MpeI was comparable to the activity of wild-type M.MpeI. 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.

Beta class amino methyltransferases from bacteria to humans: evolution and structural consequences

“…Early analysis, of the amino acid sequences of 13 bacterial DNA methyltransferases (MTases) generating 5-methylcytosine (5mC), revealed a set of ten conserved blocks of amino acid residues ( 1 ). These conserved motifs, numbered I to X from amino to carboxyl end, were found to have a constant linear order, simplifying their identification in protein sequences (particularly for the shorter or less-conserved motifs), though one alternative permutation of 5mC MTase motif order was later found ( 2 , 3 ). This aided in the discovery of mammalian 5mC-generating DNA methyltransferases Dnmt1 ( 4 ) and Dnmt3 ( 5 ).…”

“…While the MTase families differ in motif order, their structures are remarkably similar, comprising a seven-stranded β sheet (1-to-7) with a central topological switch-point between strands β1 and β4, and a characteristic reversed β hairpin (β6 and β7) at one end of the sheet next to strand β5 (Figure 2A and B ). This topology allows for circular permutation, where the same structure simply has the break between its amino beginning and carboxyl end at different points ( 38 ) and, in fact, it is possible to circularly permute a MTase in the laboratory with retention of function ( 3 ). The two most significant motifs – motif I (FxGxG) for binding SAM and motif IV (DPPY) for binding substrate Ade – juxtapose the target N6 atom of Ade in line with the methyl group and sulfur atom of SAM for catalysis, and are positioned at the carboxyl ends of the two parallel neighboring strands β1 and β4 (Figure 2C ).…”

Conversion of the CG specific M.MpeI DNA methyltransferase into an enzyme predominantly methylating CCA and CCC sites

“…We chose M.MpeI because it is structurally better characterized than M.SssI: for M.MpeI we have an X-ray structure of the specific recognition complex formed by the enzyme and cognate DNA ( 15 ), whereas for M.SssI only a computationally generated model is available ( 22 ). Moreover, in our hands M.MpeI was more active, more robust and easier to work with than M.SssI ( 23 ). We used a combination of rational and random mutagenesis guided by the X-ray structure of the specific M.MpeI-DNA recognition complex ( 15 ) to change the substrate preference of M.MpeI.…”

Z-DNA as a Tool for Nuclease-Free DNA Methyltransferase Assay

“…It is a more potent and active enzyme than DNMT1, another target enzyme studied in this work. Moreover, M.SssI is used to screen DNMT1 inhibitors as an alternative to DNMT1 as they are structurally analogous enzymes [ 54 , 55 , 56 ] and many DNMT1 inhibitors are shown to inhibit M.SssI and vice versa [ 57 , 58 , 59 ]. It is, however, ultimately important to study DNMT1 because it is biologically significant as it is directly related to human epigenetic functions.…”