title: 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 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.
authors.affiliation: Department of Chemistry and Institute for Biophysical Dynamics, Howard Hughes Medical Institute, The University of Chicago, 929 E. 57th Street, Chicago, IL 60637, USA.
authors.forename: Miao
authors.initials: M
authors.lastname: Yu
authors.affiliation: Department of Chemistry and Institute for Biophysical Dynamics, Howard Hughes Medical Institute, The University of Chicago, 929 E. 57th Street, Chicago, IL 60637, USA.
authors.forename: Chun-Xiao
authors.initials: CX
authors.lastname: Song
authors.affiliation: Department of Chemistry and Institute for Biophysical Dynamics, Howard Hughes Medical Institute, The University of Chicago, 929 E. 57th Street, Chicago, IL 60637, USA. Electronic address: chuanhe@uchicago.edu.
authors.forename: Chuan
authors.initials: C
authors.lastname: He
mesh_terms: D000818:Animals; D004247:DNA / Q000737:chemistry*; D019175:DNA Methylation; D003641:Deamination; D057890:Epigenomics / Q000379:methods*; D008322:Mammals / Q000235:genetics*; D008954:Models, Biological; D010432:Pentoxyl / Q000031:analogs & derivatives* / Q000737:chemistry
chemical_list: C008055:5-hydroxymethyluracil; D010432:Pentoxyl; D004247:DNA
keywords: 5-Hydroxymethyluracil; Deamination; Demethylation; Tet proteins