Mouse Tet1 Protein can Oxidize 5mC to 5hmC and 5caC on Single-stranded DNA

张良,; 于淼,; 何川,

doi:10.6023/a12090619

Cited by 6 publications

(3 citation statements)

References 23 publications

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“…The activity of the C-terminal catalytic domain of mTET1CD on hemiDNA and ssDNA has been reported previously (11). Here we compare the activity of mTET1CD on ssDNA, dsDNA, and gDNA, using an LC-MSbased activity assay to measure the amount of 5m C, 5hm C, 5f C, and 5ca C after a 1-h incubation.…”

Section: Resultsmentioning

confidence: 69%

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

Pais

Dai

Tamanaha

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Resultsmentioning

confidence: 69%

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

Pais

Dai

Tamanaha

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Additionally, CcTET could also oxidize 5mC in single-stranded DNA (ssDNA) with similar oxidation activity and product contents as dsDNA ( Figure S6 ). 40 In contrast, the catalytic domain of mouse TET1 (mTET1) also exhibits higher activity at pH 5.8 and 7.0 than at pH 8.0 at 37 °C. mTET1 converts almost all 5mC to 5caC under slightly acidic and neutral conditions as well as under pH 8.0 with ATP in vitro ( Figure S7 ).…”

mentioning

confidence: 99%

A TET Homologue Protein from Coprinopsis cinerea (CcTET) That Biochemically Converts 5-Methylcytosine to 5-Hydroxymethylcytosine, 5-Formylcytosine, and 5-Carboxylcytosine

et al. 2014

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DNA methylation (5-methylcytosine, 5mC) plays critical biological functions in mammals and plants as a vital epigenetic marker. The Ten-Eleven translocation dioxygenases (TET1, 2, and 3) have been found to oxidize 5mC to 5-hydroxymethylcytosine (5hmC) and then to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) in mammalian cells. We report herein three mushroom TET homologues from Coprinopsis cinerea that can mediate 5mC oxidation. Specifically, one homologue (CC1G_05589, CcTET) shows similar activity to its mammalian TET homologues. Biochemically, CcTET actively converts 5mC to 5hmC, 5fC, and 5caC under natural conditions (pH 7.0). Interestingly, CcTET also converts the majority of 5mC to 5fC under slightly acidic (pH 5.8) and neutral conditions. Kinetics analyses of the oxidation by CcTET under neutral conditions indicate that conversion of 5mC to 5hmC and 5hmC to 5fC are faster than that of 5fC to 5caC, respectively. Our results provide an example of a TET homologue in a non-mammalian organism that exhibits full 5mC-to-5caC oxidation activity and a slight preference to producing 5fC. The preferential accumulation of 5fC in the in vitro oxidation reactions under both neutral and acidic conditions may have biological implications for 5mC oxidation in fungi species.

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“…TET proteins are also known to oxidize 5mC on single-stranded DNA, , although with lower activity compared to 5mC on double-stranded DNA. TET3 is known to also exist in the cytoplasm of mammalian cells.…”

Section: Tet Oxidation and Dna Demethylationmentioning

confidence: 99%

TET Family Proteins: Oxidation Activity, Interacting Molecules, and Functions in Diseases

Zhao

2015

Chem. Rev.

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100

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Mouse Tet1 Protein can Oxidize 5mC to 5hmC and 5caC on Single-stranded DNA

Cited by 6 publications

References 23 publications

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

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

A TET Homologue Protein from Coprinopsis cinerea (CcTET) That Biochemically Converts 5-Methylcytosine to 5-Hydroxymethylcytosine, 5-Formylcytosine, and 5-Carboxylcytosine

TET Family Proteins: Oxidation Activity, Interacting Molecules, and Functions in Diseases

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