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

Abstract

Modified DNA bases in mammalian genomes, such as 5-methylcytosine (5mC) and its oxidized forms, are implicated in important epigenetic regulation processes. In human or mouse, successive enzymatic conversion of 5mC to its oxidized forms is carried out by the ten-eleven translocation (TET) proteins. Previously we reported the structure of a TET-like 5mC 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 5mC (major activity) and thymidine (T) (minor activity) in all DNA forms tested, and provide unprecedented evidence for the formation of 5-formyluridine (5fU) and 5-carboxyuridine (5caU) in vitro. Mutagenesis studies reveal a delicate balance between choice of 5mC or T as the preferred substrate. Furthermore, our results suggest substrate preference by NgTET1 to 5mCpG 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 5mC sequencing technologies such as single molecule, real-time sequencing to map 5mC in bacterial genomes at base resolution.