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 (C-5m) and its oxidized forms, are implicated in important epigenetic regulation processes. In human or mouse, successive enzymatic conversion of C-5m to its oxidized forms is carried out by the ten-eleven translocation (TET) proteins. Previously we reported the structure of a TET-like C-5m 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 C-5m (major activity) and thymidine (T) (minor activity) in all DNA forms tested, and provide unprecedented evidence for the formation of 5-formyluridine (U-5f) and 5-carboxyuridine (U-5ca) in vitro. Mutagenesis studies reveal a delicate balance between choice of C-5m 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 C-5m sequencing technologies such as single molecule, real-time sequencing to map C-5m in bacterial genomes at base resolution.