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Detection of mismatched 5-hydroxymethyluracil in DNA by selective chemical labeling.

ABSTRACT: How DNA demethylation is achieved in mammals is still under extensive investigation. One proposed mechanism is deamination of 5-hydroxymethylcytosine to form 5-hydroxymethyluracil (5hmU), followed by base excision repair to replace the mismatched 5hmU with cytosine. In this process, 5hmU:G mispair serves as a key intermediate and its localization and distribution in mammalian genome could be important information to investigate the proposed pathway. Here we describe a selective labeling method to map mismatched 5hmU. After converting other cytosine modifications to 5-carboxylcytosines, a biotin tag is installed onto mismatched 5hmU through ?-glucosyltransferase-catalyzed glucosylation and click chemistry. The enriched 5hmU-containing DNA fragments can be subject to subsequent sequencing to reveal the distribution of 5hmU:G mispair with base-resolution information acquired.

SUBMITTER: Yu M

PROVIDER: S-EPMC4300242 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Detection of mismatched 5-hydroxymethyluracil in DNA by selective chemical labeling.

Yu Miao M Song Chun-Xiao CX He Chuan C

Methods (San Diego, Calif.) 20141115

How DNA demethylation is achieved in mammals is still under extensive investigation. One proposed mechanism is deamination of 5-hydroxymethylcytosine to form 5-hydroxymethyluracil (5hmU), followed by base excision repair to replace the mismatched 5hmU with cytosine. In this process, 5hmU:G mispair serves as a key intermediate and its localization and distribution in mammalian genome could be important information to investigate the proposed pathway. Here we describe a selective labeling method t ...[more]

PMID: 25462560

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