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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 β-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.

Original publication

DOI

10.1016/j.ymeth.2014.11.007

Type

Journal article

Journal

Methods

Publication Date

15/01/2015

Volume

72

Pages

16 - 20

Keywords

5-Hydroxymethyluracil, Deamination, Demethylation, Tet proteins, Animals, DNA, DNA Methylation, Deamination, Epigenomics, Mammals, Models, Biological, Pentoxyl