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In prokaryotes, DNA methyltransferases (MTases) are part of the restriction-modification (RM) systems that, along with restriction enzymes, protect the genome against exogenous sources of DNA, such as viral infection. Here Blow et al. use deep single-molecule real-time (SMRT) sequencing to analyse the genome-wide methylation patterns across a wide range of prokaryotic species.

The authors used a panel of 217 bacterial and 13 archaeal species, selected to maximize phylogenetic diversity, and performed SMRT sequencing to identify the locations of DNA methylation and the probable type of modification: 6-methyladenosine (m6A), 4-methylcytosine (m4C) or 5-methylcytosine (m5C). The team was able to identify reccurring sequence motifs associated with methylation in each genome, which are likely to be recognition motifs for an MTase. Combined with the identification of candidate MTase genes, the group could predict the MTase that recognized each identified motif, as well as an associated restriction endonuclease (REase).

Although the authors acknowledge potential problems with genetic drift and misidentification of RNA targeting enzymes, their analysis supports existing work on the genomic organization of RM systems and goes on to identify 148 previously undescribed systems, including type II RM systems with an uncharacteristic separation of REase and MTase in the genome.

Prior work has ascribed the self-protection against type I RM systems to the m6A modification; the group here adds to this work by describing protection mediated by m4C. In these novel protection modifications, two MTase genes are associated with the RM system, with each responsible for a different modification. The authors' analysis also revealed 165 'orphan' MTases that have been evolutionarily conserved but have no known function. These orphans are associated with unmethylated motifs found in prokaryotic gene regulatory regions, including transcription start sites and sites of origin of replication.

By analysing the methylation patterns in a diverse set of prokaryotes, Blow and colleagues have shed light on the importance and conservation of these regulatory systems. They suggest that the extensive methylation seen in a range of organisms hints at an early origin for DNA modification, initially as a regulatory mechanism that was then adapted for a defensive role.