Phage-encoded ten-eleven translocation dioxygenase (TET) is active in C5-cytosine hypermodification in DNA

TET/JBP (ten-eleven translocation/base J binding protein) enzymes are iron(II)-and 2-oxo-glutarate-dependent dioxygenases that are found in all kingdoms of life and oxidize 5-methylpyrimidines on the polynucleotide level. Despite their prevalence, few examples have been biochemically characterized. Among those studied are the metazoan TET enzymes that oxidize 5-methylcytosine in DNA to hydroxy, formyl, and carboxy forms and the euglenozoa JBP diox-ygenases that oxidize thymine in the first step of base J biosynthesis. Both enzymes have roles in epigenetic regulation. It has been hy-pothesized that all TET/JBPs have their ancestral origins in bacterio-phages, but only eukaryotic orthologs have been described. Here we demonstrate the 5mC-dioxygenase activity of several phage TETs encoded within viral metagenomes. The clustering of these TETs in a phylogenetic tree correlates with the sequence specificity of their genomically cooccurring cytosine C5-methyltransferases, which in-stall the methyl groups upon which TETs operate. The phage TETs favor Gp5mC dinucleotides over the 5mCpG sites targeted by the eukaryotic TETs and are found within gene clusters specifying com-plex cytosine modifications that may be important for DNA packag-and evasion of host restriction.