title: 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, real-time sequencing to map (5m)C in bacterial genomes at base resolution.
authors.affiliation: Research Department, New England Biolabs, Ipswich, MA 01938; and.
authors.forename: June E
authors.initials: JE
authors.lastname: Pais
authors.affiliation: Research Department, New England Biolabs, Ipswich, MA 01938; and.
authors.forename: Nan
authors.initials: N
authors.lastname: Dai
authors.affiliation: Research Department, New England Biolabs, Ipswich, MA 01938; and.
authors.forename: Esta
authors.initials: E
authors.lastname: Tamanaha
authors.affiliation: Research Department, New England Biolabs, Ipswich, MA 01938; and.
authors.forename: Romualdas
authors.initials: R
authors.lastname: Vaisvila
authors.affiliation: Research Department, New England Biolabs, Ipswich, MA 01938; and.
authors.forename: Alexey I
authors.initials: AI
authors.lastname: Fomenkov
authors.affiliation: Research Department, New England Biolabs, Ipswich, MA 01938; and.
authors.forename: Jurate
authors.initials: J
authors.lastname: Bitinaite
authors.affiliation: Research Department, New England Biolabs, Ipswich, MA 01938; and.
authors.forename: Zhiyi
authors.initials: Z
authors.lastname: Sun
authors.affiliation: Research Department, New England Biolabs, Ipswich, MA 01938; and.
authors.forename: Shengxi
authors.initials: S
authors.lastname: Guan
authors.affiliation: Research Department, New England Biolabs, Ipswich, MA 01938; and.
authors.forename: Ivan R
authors.initials: IR
authors.lastname: Corrêa
authors.affiliation: Research Department, New England Biolabs, Ipswich, MA 01938; and.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Noren
authors.affiliation: Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322.
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Cheng
authors.affiliation: Research Department, New England Biolabs, Ipswich, MA 01938; and.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Roberts
authors.affiliation: Research Department, New England Biolabs, Ipswich, MA 01938; and saleh@neb.com yu.zhengyu@gmail.com.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zheng
authors.affiliation: Research Department, New England Biolabs, Ipswich, MA 01938; and saleh@neb.com yu.zhengyu@gmail.com.
authors.forename: Lana
authors.initials: L
authors.lastname: Saleh
mesh_terms: D044503:5-Methylcytosine / Q000737:chemistry*; D000465:Algorithms; D000818:Animals; D003596:Cytosine / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry; D044127:Epigenesis, Genetic; D057890:Epigenomics; D006801:Humans; D051379:Mice; D006899:Mixed Function Oxygenases / Q000737:chemistry; D009154:Mutation; D009253:Naegleria / Q000201:enzymology*; D010100:Oxygen / Q000737:chemistry; D010105:Oxygenases / Q000737:chemistry*; D010802:Phylogeny; D011518:Proto-Oncogene Proteins / Q000737:chemistry; D015800:Protozoan Proteins / Q000737:chemistry*; D017422:Sequence Analysis, DNA; D013936:Thymidine / Q000737:chemistry
chemical_list: D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D015800:Protozoan Proteins; D044503:5-Methylcytosine; D003596:Cytosine; D004247:DNA; D006899:Mixed Function Oxygenases; C462539:TET1 protein, human; D010105:Oxygenases; D010100:Oxygen; D013936:Thymidine
keywords: 5-methylcytosine; NgTET1; SMRT sequencing; TET proteins; bacterial methylome
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