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Author Notes:

To whom correspondence should be addressed. Tel: +1 404 727 8491; Fax: +1 404 727 3746; Email: xcheng@emory.edu

H.H. performed all experiments with hTDG, and eMUG.

S.H. performed experiments with plant ROS1.

A.S.B. provided purified eMUG and help in editing the manuscript.

X. Z. and X.C. organized and designed the scope of the study, and all were involved in analyzing data and preparing the manuscript.

We thank sincerely Brenda Baker of New England Biolabs for DNA oligosynthesis, Drs John R. Horton and John Chrzas for help with X-ray data collections, Dr Jian-Kang Zhu for providing ROS1 expression vector and Dr Robert M. Blumenthal of the University of Toledo College of Medicine for critical comments and editing the manuscript.

Conflict of interest statement. None declared.

Subject:

Research Funding:

U.S. National Institutes of Health (GM049245-18; to X.C. and GM057200-09 to A.S.B.); Georgia Research Alliance Eminent Scholar (to X.C.) and the Department of Biochemistry at the Emory University School of Medicine (use of the Southeast Regional Collaborative Access Team synchrotron beamlines at the Advanced Photon Source of Argonne National Laboratory).

Funding for open access charge: NIH.

Keywords:

  • Science & Technology
  • Life Sciences & Biomedicine
  • Biochemistry & Molecular Biology
  • CRYSTAL-STRUCTURE
  • SUBSTRATE-SPECIFICITY
  • MISMATCH RECOGNITION
  • ESCHERICHIA-COLI
  • MAMMALIAN DNA
  • REPAIR ENZYME
  • 5-METHYLCYTOSINE
  • URACIL
  • 5-FORMYLCYTOSINE
  • KINETICS

Excision of 5-hydroxymethyluracil and 5-carboxylcytosine by the thymine DNA glycosylase domain: its structural basis and implications for active DNA demethylation

Journal Title:

Nucleic Acids Research

Volume:

Volume 40, Number 20

Publisher:

, Pages 10203-10214

Type of Work:

Article | Final Publisher PDF

Abstract:

The mammalian thymine DNA glycosylase (TDG) is implicated in active DNA demethylation via the base excision repair pathway. TDG excises the mismatched base from G:X mismatches, where X is uracil, thymine or 5-hydroxymethyluracil (5hmU). These are, respectively, the deamination products of cytosine, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). In addition, TDG excises the Tet protein products 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) but not 5hmC and 5mC, when paired with a guanine. Here we present a post-reactive complex structure of the human TDG domain with a 28-base pair DNA containing a G:5hmU mismatch. TDG flips the target nucleotide from the double-stranded DNA, cleaves the N-glycosidic bond and leaves the C1′ hydrolyzed abasic sugar in the flipped state. The cleaved 5hmU base remains in a binding pocket of the enzyme. TDG allows hydrogen-bonding interactions to both T/U-based (5hmU) and C-based (5caC) modifications, thus enabling its activity on a wider range of substrates. We further show that the TDG catalytic domain has higher activity for 5caC at a lower pH (5.5) as compared to the activities at higher pH (7.5 and 8.0) and that the structurally related Escherichia coli mismatch uracil glycosylase can excise 5caC as well. We discuss several possible mechanisms, including the amino-imino tautomerization of the substrate base that may explain how TDG discriminates against 5hmC and 5mC. © 2012 The Author(s).

Copyright information:

© 2012 Hashimoto et al. Published by Oxford University Press

This is an Open Access work distributed under the terms of the Creative Commons Attribution 3.0 Unported License (http://creativecommons.org/licenses/by/3.0/).

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