Affinity photo-crosslinking study of the DNA base flipping pathway by HhaI methyltransferase

Nucleic Acids Symp Ser. 2000:(44):271-2. doi: 10.1093/nass/44.1.271.

Abstract

HhaI DNA methyltransferase flips the inner cytosine in the recognition sequence 5'-GCGC-3' out of the DNA helix and into the catalytic site for methylation. To identify intermediate states on the base-flipping pathway, affinity photo-crosslinking experiments were performed with synthetic dodecamer duplexes containing modified bases 2-thiothymine (2sT) or 4-thiothymine (4sT) at the target base position. Here we show that the DNA strand containing 2sT, but not 4sT, covalently cross-links to the HhaI methyltransferase upon irradiation at 340-360 nm.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Affinity Labels
  • Base Sequence
  • Catalytic Domain
  • Cross-Linking Reagents
  • DNA / chemistry*
  • DNA / metabolism*
  • DNA / radiation effects
  • DNA Methylation
  • DNA-Cytosine Methylases*
  • In Vitro Techniques
  • Oligodeoxyribonucleotides / chemistry
  • Oligodeoxyribonucleotides / metabolism
  • Photochemistry
  • Thymidine / analogs & derivatives
  • Thymidine / chemistry
  • Thymine / analogs & derivatives*
  • Thymine / chemistry

Substances

  • Affinity Labels
  • Cross-Linking Reagents
  • Oligodeoxyribonucleotides
  • 2-thiothymine
  • DNA
  • DNA modification methylase HhaI
  • DNA-Cytosine Methylases
  • Thymine
  • Thymidine