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Author Notes:

Correspondence: saleh@neb.com or yu.zhengyu@gmail.com

Author contributions: J.E.P., C.J.N., X.C., R.J.R., Y.Z., and L.S. designed research; J.E.P., E.T., R.V., A.I.F., J.B., and L.S. performed research; S.G. contributed new reagents/analytic tools; J.E.P., N.D., E.T., R.V., A.I.F., Z.S., I.R.C., Y.Z., and L.S. analyzed data; and J.E.P., Y.Z., and L.S. wrote the paper.

We thank Derrick Xu and Meg Mabuchi for initial work on the project; Rick Morgan and Yvette Luyten for providing the pRS(M.HpaII) plasmid and M.Fnu4HI gDNA; Chandler Fulton and Elaine Lai for helpful discussions; and Bill Jack for critical review of the manuscript.

The authors declare no conflict of interest.

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Research Funding:

This work is supported by New England Biolabs, and by National Institutes of Health Grants GM105132 (to Y.Z.) and GM049245-21 (to X.C.). Funding for the open access charge is from New England Biolabs.

Keywords:

  • Science & Technology
  • Multidisciplinary Sciences
  • Science & Technology - Other Topics
  • TET proteins
  • NgTET1
  • 5-methylcytosine
  • SMRT sequencing
  • bacterial methylome
  • Complex modifications
  • Coprinopsis-cinerea
  • Helicobacter
  • Mammalian DNA
  • 5-Hydroxymethylcystosine
  • 5-Carboxylcystosine
  • Oxidation
  • Proteins
  • 5MC
  • 5-Formylcytosine

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

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Journal Title:

PNAS

Volume:

Volume 112, Number 14

Publisher:

, Pages 4316-4321

Type of Work:

Article | Final Publisher PDF

Abstract:

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

Copyright information:

© 2015 National Academy of Sciences.

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