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 ( 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, realtime sequencing to map 5m C in bacterial genomes at base resolution.odified DNA bases exist in all forms of life, from viruses to mammals with many different biological roles. Accordingly, diverse mechanisms have evolved to "write," "read," and "erase" these modifications. In mammals, 5-methylcytosine ( 5m C) is the major form of DNA modification and is implicated in many crucial developmental processes. In human and mouse, 5m C can be successively oxidized into 5-hydroxymethylcytosine ( 5hm C), 5-formylcytosine ( 5f C), and 5-carboxylcytosine ( 5ca C) by the teneleven translocation (TET) family of oxygenases (1-4). The bases of 5f C and 5ca C can be excised by thymine DNA glycosylase (4). The 5m C-oxidation-coupled base-excision repair pathway provides a plausible route for active demethylation in mammalian cells. Many other species, from simple to complex, maintain DNA methylation machinery throughout their life cycle that may contribute to epigenetic regulation. Therefore, an interesting perspective is to examine shared and distinct features of TET oxygenases in diverse eukaryotes (5, 6).The human and mouse genomes encode three paralogous TET proteins, TET1, TET2, and TET3, which presumably carry out both redundant and distinct functions (7,8). TET proteins belong to the diverse group of α-ketoglutarate (αKG) and Fe(II)-dependent oxygenases (5). Subgroup classification based on sequence similarity links the TET proteins to base J-binding proteins (JBP1 and JBP2), which are primarily present in trypanosomes and possess thymidine (T)-hydroxylation activity (1). Further bioinformatic analysis revealed eight paralogous TET/ JBP-like genes in the genome of Naegleria gruberi, a single-celled amoeboflagellate protist that is a distant cousin of the par...

Adenine methylation in eukaryotes: Apprehending the complex evolutionary history and functional potential of an epigenetic modification

“…DmTET is closely related to vertebrate TETs, and retains all features of the active site of the latter enzymes which accommodate a pyrimidine rather than a purine 15, 49. A diversity of characterized eukaryotic TET/JBP enzymes have been observed only to modify 5‐methylpyrimidines rather than purines 49, 50, 51, 104, 105. This is further confirmed by contextual analysis of bacterial and phage TET/JBP family members, which are predicted to primarily modify pyrimidines 15, 90.…”

Sequencing 5‐Hydroxymethyluracil at Single‐Base Resolution

“…[1] 5-hydroxymethyluracil (5hmU) is produced through oxidation of thymine both enzymatically and non-enzymatically [2] and can influence the binding of proteins to DNA. [2b,3] It has also been suggested that 5hmU might lead to genomic instability as it can be removed by DNAr epair enzymes to create potentially mutagenic lesions [4] and affect the stability of DNA duplexes.…”

The Mechanisms of Generation, Recognition, and Erasure of DNA 5-Methylcytosine and Thymine Oxidations

“…Very recently, it has been shown that Tet enzymes are also active on thymine, generating 5-hydroxymethyluracil (5hmU) paired with adenine that could be specifically bound by potential protein readers (22,23) (Fig. 1f).…”

“…Perhaps TpG dinucleotides are selected for when it is advantageous for a particular DNA sequence to be treated as if it were permanently (hemi)-methylated. Further modification by Tet enzymes on both 5mC and T would generate fully modified CpA/TpG sites with 5-hydroxymethyl or even 5-formyl and 5-carboxyl modification on both 5mC and T (23). Such fully modified CpA/TpG might even be maintained during DNA replication through the combined activity of Dnmt3 and Tet enzymes (Fig.…”

“…In light of the recent observation that 5hmU could also be the product of Tet-mediated thymine hydroxylation (22,23), it is interesting to note that SMUG1 (named after single-strandspecific monofunctional uracil-DNA glycosylase) can excise 5hmU when mispaired with a guanine as well as paired with an adenine (86) or 5fU in any base pair context (87). In addition, members of the NEIL family of DNA glycosylases have been found to bind oxidized 5mC derivatives 5hmC, 5fC, or 5caC (88) and 5hmU (22), and partially rescue the loss of TDG in a gene reactivation assay (89).…”