DNA methyltransferase probing of DNA-protein interactions

Methods Mol Biol. 2006:338:225-44. doi: 10.1385/1-59745-097-9:225.

Abstract

Effective methods of probing chromatin structure without disrupting DNA-protein interactions and associations are necessary for creating an accurate picture of chromatin and its processes in vivo. Expression of cytidine-5 DNA methyltransferases (C5 DMTases) in Saccharomyces cerevisiae provides a powerful noninvasive method of assaying relative DNA accessibility in chromatin. DNA MTases are occluded from protein-associated DNA based on the strength and span of the DNA-protein interaction. Ectopic regulation of C5 DMTase expression systems allows for minimal disruption of yeast physiology. Methylated sites are detected by bisulfite genomic sequencing, which leads to a positive signal corresponding to modified cytidine residues. High-resolution C5 DMTases with dinucleotide recognition specificity are shown to provide sufficient coverage to map interactions spanning a relatively short distance.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Chromatin / genetics
  • Chromatin / metabolism
  • DNA (Cytosine-5-)-Methyltransferases* / genetics
  • DNA (Cytosine-5-)-Methyltransferases* / metabolism
  • DNA / genetics
  • DNA / metabolism*
  • DNA Primers / genetics
  • DNA, Fungal / genetics
  • DNA, Fungal / metabolism
  • Escherichia coli / genetics
  • Molecular Probe Techniques*
  • Nuclear Proteins / metabolism*
  • Polymerase Chain Reaction
  • Protein Binding
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Transformation, Genetic

Substances

  • Chromatin
  • DNA Primers
  • DNA, Fungal
  • Nuclear Proteins
  • Recombinant Proteins
  • DNA
  • DNA (Cytosine-5-)-Methyltransferases