Biochemical characterization of ParI, an orphan C5-DNA methyltransferase from Psychrobacter arcticus 273-4

Abstract

Cytosine-specific DNA methyltransferases are important enzymes in most living organisms. In prokaryotes, most DNA methyltransferases are members of the type II restriction-modification system where they methylate host DNA, thereby protecting it from digestion by the accompanying restriction endonucleases. DNA methyltransferases can also act as solitary enzymes having important roles in controlling gene expression, DNA replication, cell cycle and DNA post-replicative mismatch repair. They have potential applications in biotechnology, such as in labeling of biopolymers, DNA mapping or epigenetic analysis, as well as for general DNA-protein interaction studies. The parI gene from the psychrophilic bacterium Psychrobacter arcticus 273-4 encodes a cytosine-specific DNA methyltransferase. In this work, recombinant ParI was expressed and purified in fusion to either an N-terminal hexahistidine affinity tag, or a maltose binding protein following the hexahistidine affinity tag, for solubility improvement. After removal of the fusion partners, recombinant ParI was found to be monomeric by size exclusion chromatography, with its molecular mass estimated to be 54 kDa. The apparent melting temperature of the protein was 53 °C with no detectable secondary structures above 65 °C. Both recombinant and native ParI showed methyltransferase activity in vivo. In addition, MBP- and His-tagged ParI also demonstrated in vitro activity. Although the overall structure of ParI exhibits high thermal stability, the loss of in vitro activity upon removal of solubility tags or purification from the cellular milieu indicates that the catalytically active form is more labile. Horizontal gene transfer may explain the acquisition of a protein-encoding gene that does not display common cold-adapted features.

An Inducible Microbacterium Prophage vB_MoxS-R1 Represents a Novel Lineage of Siphovirus

“…DNA methylation is an important DNA modification [95]. DNA methyltransferases play vital roles in many cellular processes, such as DNA replication, protection, post-replicative mismatch repair, protein-DNA interaction and cell cycle [96][97][98]. Most prokaryotic DNA methyltransferases belong to the restriction-modification system [97].…”

“…DNA methyltransferases play vital roles in many cellular processes, such as DNA replication, protection, post-replicative mismatch repair, protein-DNA interaction and cell cycle [96][97][98]. Most prokaryotic DNA methyltransferases belong to the restriction-modification system [97]. Methyltransferases can protect specific DNA sequences from digestion by restriction endonucleases [97,99].…”

“…Most prokaryotic DNA methyltransferases belong to the restriction-modification system [97]. Methyltransferases can protect specific DNA sequences from digestion by restriction endonucleases [97,99]. In addition, cytosine-specific DNA methylase plays a crucial role in viral maturation in infected Escherichia coli cells [100].…”