Excision of thymine and 5-hydroxymethyluracil by the MBD4 DNA glycosylase domain: structural basis and implications for active DNA demethylation

Excision of thymine and 5-hydroxymethyluracil by the MBD4 DNA glycosylase domain: structural basis and implications for active DNA demethylation

The mammalian DNA glycosylase—methyl-CpG binding domain protein 4 (MBD4)—is involved in active DNA demethylation via the base excision repair pathway. MBD4 contains an N-terminal MBD and a C-terminal DNA glycosylase domain. MBD4 can excise the mismatched base paired with a guanine (G:X), where X is...

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Main Authors: Hashimoto, Hideharu, Zhang, Xing, Cheng, Xiaodong
Format: Online
Language:English
Published: Oxford University Press 2012
Online Access:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3458566/
id pubmed-3458566
recordtype oai_dc
spelling pubmed-34585662012-09-27 Excision of thymine and 5-hydroxymethyluracil by the MBD4 DNA glycosylase domain: structural basis and implications for active DNA demethylation Hashimoto, Hideharu Zhang, Xing Cheng, Xiaodong Gene Regulation, Chromatin and Epigenetics The mammalian DNA glycosylase—methyl-CpG binding domain protein 4 (MBD4)—is involved in active DNA demethylation via the base excision repair pathway. MBD4 contains an N-terminal MBD and a C-terminal DNA glycosylase domain. MBD4 can excise the mismatched base paired with a guanine (G:X), where X is uracil, thymine or 5-hydroxymethyluracil (5hmU). These are, respectively, the deamination products of cytosine, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Here, we present three structures of the MBD4 C-terminal glycosylase domain (wild-type and its catalytic mutant D534N), in complex with DNA containing a G:T or G:5hmU mismatch. MBD4 flips the target nucleotide from the double-stranded DNA. The catalytic mutant D534N captures the intact target nucleotide in the active site binding pocket. MBD4 specifically recognizes the Watson–Crick polar edge of thymine or 5hmU via the O2, N3 and O4 atoms, thus restricting its activity to thymine/uracil-based modifications while excluding cytosine and its derivatives. The wild-type enzyme cleaves the N-glycosidic bond, leaving the ribose ring in the flipped state, while the cleaved base is released. Unexpectedly, the C1′ of the sugar has yet to be hydrolyzed and appears to form a stable intermediate with one of the side chain carboxyl oxygen atoms of D534, via either electrostatic or covalent interaction, suggesting a different catalytic mechanism from those of other DNA glycosylases. Oxford University Press 2012-09 2012-06-27 /pmc/articles/PMC3458566/ /pubmed/22740654 http://dx.doi.org/10.1093/nar/gks628 Text en © The Author(s) 2012. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/3.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
repository_type Open Access Journal
institution_category Foreign Institution
institution US National Center for Biotechnology Information
building NCBI PubMed
collection Online Access
language English
format Online
author Hashimoto, Hideharu
Zhang, Xing
Cheng, Xiaodong
spellingShingle Hashimoto, Hideharu
Zhang, Xing
Cheng, Xiaodong
Excision of thymine and 5-hydroxymethyluracil by the MBD4 DNA glycosylase domain: structural basis and implications for active DNA demethylation
author_facet Hashimoto, Hideharu
Zhang, Xing
Cheng, Xiaodong
author_sort Hashimoto, Hideharu
title Excision of thymine and 5-hydroxymethyluracil by the MBD4 DNA glycosylase domain: structural basis and implications for active DNA demethylation
title_short Excision of thymine and 5-hydroxymethyluracil by the MBD4 DNA glycosylase domain: structural basis and implications for active DNA demethylation
title_full Excision of thymine and 5-hydroxymethyluracil by the MBD4 DNA glycosylase domain: structural basis and implications for active DNA demethylation
title_fullStr Excision of thymine and 5-hydroxymethyluracil by the MBD4 DNA glycosylase domain: structural basis and implications for active DNA demethylation
title_full_unstemmed Excision of thymine and 5-hydroxymethyluracil by the MBD4 DNA glycosylase domain: structural basis and implications for active DNA demethylation
title_sort excision of thymine and 5-hydroxymethyluracil by the mbd4 dna glycosylase domain: structural basis and implications for active dna demethylation
description The mammalian DNA glycosylase—methyl-CpG binding domain protein 4 (MBD4)—is involved in active DNA demethylation via the base excision repair pathway. MBD4 contains an N-terminal MBD and a C-terminal DNA glycosylase domain. MBD4 can excise the mismatched base paired with a guanine (G:X), where X is uracil, thymine or 5-hydroxymethyluracil (5hmU). These are, respectively, the deamination products of cytosine, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Here, we present three structures of the MBD4 C-terminal glycosylase domain (wild-type and its catalytic mutant D534N), in complex with DNA containing a G:T or G:5hmU mismatch. MBD4 flips the target nucleotide from the double-stranded DNA. The catalytic mutant D534N captures the intact target nucleotide in the active site binding pocket. MBD4 specifically recognizes the Watson–Crick polar edge of thymine or 5hmU via the O2, N3 and O4 atoms, thus restricting its activity to thymine/uracil-based modifications while excluding cytosine and its derivatives. The wild-type enzyme cleaves the N-glycosidic bond, leaving the ribose ring in the flipped state, while the cleaved base is released. Unexpectedly, the C1′ of the sugar has yet to be hydrolyzed and appears to form a stable intermediate with one of the side chain carboxyl oxygen atoms of D534, via either electrostatic or covalent interaction, suggesting a different catalytic mechanism from those of other DNA glycosylases.
publisher Oxford University Press
publishDate 2012
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3458566/
_version_ 1611911952310730752

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