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. 2015 Apr 7;112(14):4316-21.
doi: 10.1073/pnas.1417939112. Epub 2015 Mar 23.

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

June E Pais  1 , Nan Dai  1 , Esta Tamanaha  1 , Romualdas Vaisvila  1 , Alexey I Fomenkov  1 , Jurate Bitinaite  1 , Zhiyi Sun  1 , Shengxi Guan  1 , Ivan R Corrêa Jr  1 , Christopher J Noren  1 , Xiaodong Cheng  2 , Richard J Roberts  1 , Yu Zheng  3 , Lana Saleh  3
Affiliations

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

June E Pais et al. Proc Natl Acad Sci U S A. .

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.

Keywords: 5-methylcytosine; NgTET1; SMRT sequencing; TET proteins; bacterial methylome.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Enzymatic activity of NgTET1 on oligo, plasmid and gDNA. (A) RE-based assay showing protection of NgTET1-treated pRS(M.HpaII) plasmid against digestion with MspI at varying concentrations of NgTET1. (B) LC-MS (Agilent 1200)-based assay reflecting NgTET1 reaction species using mammalian gDNA IMR90. Reactions contained 1.5 μg sheared (1.5-kb) DNA and 4 μM NgTET1. (C–E) Quantification of NgTET1 reaction species as measured by LC-MS (Agilent 1200 for C and D; 6490 Triple Quad LC-MS for E) for different types of DNA. The error bars (in black) represent the SEM (n ≥ 3). (C) Two micromolars oligo (Table S1), 1.5 μg plasmid, and 1.5 μg gDNA were used with 4 µM NgTET1. (D) Four micromolars 5mC sites for symmDNA, hemiDNA and ssDNA (Table S1) were used with 8 μM NgTET1. (E) Two micromolars ds- or ss-oligo (Table S1) or sheared (1.5-kb) HeLa (0.5 μg) or M.Fnu4HI (0.2 μg) gDNA were used with 6.7 µM NgTET1 (in Mops buffer pH 6.9) or mTET1CD.
Fig. 2.
Fig. 2.
T-oxygenase activity of NgTET1. (A) Kinetic time course depicting the decay of 5mC or T for a reaction with 4 μM NgTET1 and 2 μM oligo C5mCGG. Reaction species were detected and quantified by LC-MS (Agilent 1200). The data are fit to a single exponential and the observed rate constants with SEM are provided. (B) Quantification of oxidized T reaction species using 6.7 μM mTET1CD or NgTET1 (in Mops buffer pH 6.9) as measured by LC-MS (6490 Triple Quad LC-MS) for 0.2 μg sheared (1.5-kb) gDNA substrates. The error bars (in black) represent the SE (SEM) (n ≥ 3). (C) LC-MS (Agilent 1200) traces comparing T-oxygenase activity by NgTET1 (10 μM) on methylated and unmethylated pUC19 plasmid DNA (2.5 μg). (D) LC-MS (Agilent 1200) quantification of 5mC or T after a 1-h reaction of 4 μM NgTET1 WT or variant proteins with 2 μM oligo C5mCGG. Error bars (in black) represent the SEM (n ≥ 3).
Fig. 3.
Fig. 3.
NgTET1 activity is dependent on nucleotide-sequence context. Distribution of NgTET1 reaction species: (A and B) As quantified by LC-MS (Agilent 1200), using excess enzyme (20 µM) with (A) genomic (2.5 μg, sheared to 1.5-kb) or (B) plasmid (2.5 μg) DNA containing different methylation sequences as indicated in red (Table S2); (C and D) As quantified by 6490 Triple Quad LC-MS for (C) 30-min reaction of 8 μM NgTET1 with 4 μM oligo DNA or (D) 6.7 μM NgTET1 (in Mops buffer pH 6.9) or mTET1CD with 1.6 μM oligo DNA. For A–D, error bars (in black) represent the SEM (n ≥ 3). (E) Kinetic traces, with species fraction determined by LC-MS (Agilent 1200), of NgTET1 with 5mC-, 5hmC-, or 5fC-containing oligos hemimethylated at a single CpX site (Table S1). Reactions were done in Mops buffer (pH 6.75) using 8 μM NgTET1 and 4 μM DNA. The data are fit to a single exponential and the observed rate constants with SEM are provided.
Fig. 4.
Fig. 4.
SMRT sequencing of H. pylori gDNA using NgTET1. (A) Sequence logos for 5mCCTC, 5mCCTTC and G5mCGC motifs detected by SMRT sequencing of NgTET1-treated gDNA. (B) IPD ratio plots corresponding to the 5mCCTC motif in gDNA treated in the absence of NgTET1 (Top), with NgTET1 (Middle), or with NgTET1/NaBH4/T4-βGT (Bottom). (C) IPD ratio plots for the sequences detected (Upper) versus undetected (Lower) as belonging to the G5mCGC motif for gDNA treated with NgTET1/NaBH4/T4-βGT. For B and C, the error bars (in black) represent the SEM. (D) Scatter plot of IPD ratio values at the methylated and +2 positions for G5mCGC and CCGG sequences for gDNA treated with NgTET1/NaBH4/T4-βGT. (E) Plot of sensitivity and specificity as a function of IPD ratio for gDNA treated with NgTET1/NaBH4/T4-βGT.
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