期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 83, 期 1, 页码 104-111出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2012.03.003
关键词
Arabidopsis thaliana; VIM1 (Variant in Methylation 1) protein; 5-Methyl-cytosine 5-hydroxymethyl-cytosine; Electrophoretic-mobility shift (EMSA) assay; Fluorescence anisotrophy (FA) assays
类别
资金
- Office of Biological and Environmental Research of the U.S. Department of Energy
The discovery of 5-hydroxymethyl-cytosine (5hmC) in mammalian cells prompted us to look for this base in the DNA of Arabidopsis thaliana (thale cress), and to ask how well the Arabidopsis Variant in Methylation 1 (VIM1) protein, an essential factor in maintaining 5-cytosine methylation (5mC) homeostasis and epigenetic silencing in this plant, recognizes this novel base. We found that the DNA of Arabidopsis' leaves and flowers contain low levels of 5hmC. We also cloned and expressed in Escherichia coli full-length VIM1 protein, the archetypal member of the five Arabidopsis VIM gene family. Using in vitro binding assays, we observed that full-length VIM1 binds preferentially to hemi-methylated DNA with a single modified 5mCpG site; this result is consistent with its known role in preserving DNA methylation in vivo following DNA replication. However, when 5hmC replaces one or both cytosine residues at a palindromic CpG site, VIM1 binds with approximately >= 10-fold lower affinity. These results suggest that 5hmC may contribute to VIM-mediated passive loss of cytosine methylation in vivo during Arabidopsis DNA replication. (C) 2012 Elsevier Inc. All rights reserved.
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