Biochemical characterization of a Naegleria TET-like oxygenase and its application in single molecule sequencing of 5-methylcytosine

Modified DNA bases in mammalian genomes, such as 5-methylcytosine ( 5m C) and its oxidized forms, are implicated in important epigenetic regulation processes. In human or mouse, successive enzymatic conversion of 5m C to its oxidized forms is carried out by the ten-eleven translocation (TET) proteins. Previously we reported the structure of a TET-like 5m C oxygenase (NgTET1) from Naegleria gruberi , a single-celled protist evolutionarily distant from vertebrates. Here we show that NgTET1 is a 5-methylpyrimidine oxygenase, with activity on both 5m C (major activity) and thymidine (T) (minor activity) in all DNA forms tested, and provide unprecedented evidence for the formation of 5-formyluridine ( 5f U) and 5-carboxyuridine ( 5ca U) in vitro. Mutagenesis studies reveal a delicate balance between choice of 5m C or T as the preferred substrate. Furthermore, our results suggest substrate preference by NgTET1 to 5m CpG and TpG dinucleotide sites in DNA. Intriguingly, NgTET1 displays higher T-oxidation activity in vitro than mammalian TET1, supporting a closer evolutionary relationship between NgTET1 and the base J-binding proteins from trypanosomes. Finally, we demonstrate that NgTET1 can be readily used as a tool in 5m C sequencing technologies such as single molecule, real-time sequencing to map 5m C in bacterial genomes at base resolution.