Differential stabilization of reaction intermediates: specificity checkpoints for M.EcoRI revealed by transient fluorescence and fluorescence lifetime studies

Ben Youngblood, Eleanor Bonnist, David T. F. Dryden, Anita C. Jones, Norbert O. Reich

Research output: Contribution to journalArticlepeer-review

Abstract

M.EcoRI, a bacterial sequence-specific S-adenosyl-L-methionine-dependent DNA methyltransferase, relies on a complex conformational mechanism to achieve its remarkable specificity, including DNA bending, base flipping and intercalation into the DNA. Using transient fluorescence and fluorescence lifetime studies with cognate and noncognate DNA, we have characterized several reaction intermediates involving the WT enzyme. Similar studies with a bending-impaired, enhanced-specificity M.EcoRI mutant show minimal differences with the cognate DNA, but significant differences with noncognate DNA. These results provide a plausible explanation of the way in which destabilization of reaction intermediates can lead to changes in substrate specificity.

Original languageEnglish
Pages (from-to)2917-2925
Number of pages9
JournalNucleic Acids Research
Volume36
Issue number9
DOIs
Publication statusPublished - May 2008

Keywords

  • ECORI DNA METHYLTRANSFERASE
  • TIME-RESOLVED FLUORESCENCE
  • ACTIVE-SITE
  • BASE
  • BINDING
  • 2-AMINOPURINE
  • METHYLATION
  • TARGET

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