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

• Type of Material: Article
• Authors: Hideharu Hashimoto, Emory University; Samuel Hong, Emory University; Ashok S. Bhagwat, Wayne State University; Xing Zhang, Emory University; Xiaodong Cheng, Emory University
• Language: English
• Date: 2012-11-01
• Publisher: Oxford University Press (OUP): Policy C - Option B
• Publication Version: Final Published Version
• Copyright Statement: © 2012 Hashimoto et al. Published by Oxford University Press
• License: Creative Commons Attribution 3.0 Unported
• Final Published Version (URL): http://dx.doi.org/10.1093/nar/gks845
• Title of Journal or Parent Work: Nucleic Acids Research
• ISSN: 0305-1048
• Volume: 40
• Issue: 20
• Start Page: 10203
• End Page: 10214
• Grant/Funding Information: Funding for open access charge: NIH.; 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).
• Keywords: MISMATCH RECOGNITION; MAMMALIAN DNA; Life Sciences & Biomedicine; Science & Technology; 5-FORMYLCYTOSINE; KINETICS; Biochemistry & Molecular Biology; REPAIR ENZYME; SUBSTRATE-SPECIFICITY; CRYSTAL-STRUCTURE; URACIL; ESCHERICHIA-COLI; 5-METHYLCYTOSINE
• Research Categories: Chemistry, Biochemistry

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).