Heterologous expression and purification of Arabidopsis thaliana VIM1 protein: in vitro evidence for its inability to recognize hydroxymethylcytosine, a rare base in Arabidopsis DNA

Protein Expr Purif. 2012 May;83(1):104-11. doi: 10.1016/j.pep.2012.03.003. Epub 2012 Mar 20.


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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • 5-Methylcytosine / chemistry
  • 5-Methylcytosine / metabolism
  • Arabidopsis Proteins / chemistry
  • Arabidopsis Proteins / isolation & purification
  • Arabidopsis Proteins / metabolism*
  • Cytosine / analogs & derivatives*
  • Cytosine / chemistry
  • Cytosine / metabolism
  • DNA Methylation / physiology
  • DNA, Plant / chemistry
  • DNA, Plant / metabolism*
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / isolation & purification
  • DNA-Binding Proteins / metabolism*
  • Electrophoresis, Polyacrylamide Gel
  • Electrophoretic Mobility Shift Assay
  • Escherichia coli / metabolism
  • Models, Molecular
  • Protein Binding
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism*
  • Spectrometry, Fluorescence
  • Substrate Specificity


  • Arabidopsis Proteins
  • DNA, Plant
  • DNA-Binding Proteins
  • Recombinant Proteins
  • VIM1 protein, Arabidopsis
  • 5-hydroxymethylcytosine
  • 5-Methylcytosine
  • Cytosine