Using the fluorescence decay of 2-aminopurine to investigate conformational change in the recognition sequence of the EcoRV DNA-(adenine-N6)-methyltransferase on enzyme binding

Eleanor Y. M. Bonnist, Kirsten Liebert, David T. F. Dryden, Albert Jeltsch, Anita C. Jones

Research output: Contribution to journalArticlepeer-review

Abstract

The EcoRV DNA methyltransferase methylates the first adenine in the GATATC recognition sequence. It is presumed that methylation proceeds by a nucleotide flipping mechanism but no crystal structure is available to confirm this. A popular solution-phase assay for nucleotide flipping employs the fluorescent adenine analogue, 2-aminopurine (2AP), substituted at the methylation target site; a substantial increase in fluorescence intensity on enzyme binding indicates flipping. However, this appeared to fail for M.EcoRV, since 2AP substituted for the non-target adenine in the recognition sequence showed a much greater intensity increase than 2AP at the target site. This anomaly is resolved by recording the fluorescence decay of 2AP which shows that the target 2AP is indeed flipped by the enzyme, but its fluorescence is quenched by interaction with aromatic residues in the catalytic site, whereas bending of the duplex at the non-target site alleviates inter-base quenching and exposes the 2AP to solvent. (C) 2011 Elsevier B.V. All rights reserved.

Original languageEnglish
Pages (from-to)28-34
Number of pages7
JournalBiophysical Chemistry
Volume160
Issue number1
Early online date9 Sep 2011
DOIs
Publication statusPublished - Jan 2012

Keywords

  • 2-aminopurine fluorescence
  • DNA-protein interaction
  • DNA methylation
  • Nucleotide flipping
  • Base flipping
  • TIME-RESOLVED FLUORESCENCE
  • SITE-DIRECTED MUTAGENESIS
  • N6-ADENINE DNA METHYLTRANSFERASE
  • AMINO-ACID-RESIDUES
  • SUBSTRATE-RECOGNITION
  • MOLECULAR ENZYMOLOGY
  • MUTATIONAL ANALYSIS
  • ACTIVE-SITE
  • BASE
  • METHYLATION

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