Fusion of GFP to the M.EcoKI DNA methyltransferase produces a new probe of Type I DNA restriction and modification enzymes

Kai Chen, Gareth A. Roberts, Augoustinos S. Stephanou, Laurie P. Cooper, John H. White, David T. F. Dryden

Research output: Contribution to journalArticlepeer-review

Abstract

We describe the fusion of enhanced green fluorescent protein to the C-terminus of the HsdS DNA sequence-specificity subunit of the Type I DNA modification methyltransferase M.EcoKI. The fusion expresses well in vivo and assembles with the two HsdM modification subunits. The fusion protein functions as a sequence-specific DNA methyltransferase protecting DNA against digestion by the EcoKI restriction endonuclease. The purified enzyme shows Forster resonance energy transfer to fluorescently-labelled DNA duplexes containing the target sequence and to fluorescently-labelled ocr protein, a DNA mimic that binds to the M.EcoKI enzyme. Distances determined from the energy transfer experiments corroborate the structural model of M.EcoKI. (C) 2010 Elsevier Inc. All rights reserved.

Original languageEnglish
Pages (from-to)254-259
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume398
Issue number2
DOIs
Publication statusPublished - 23 Jul 2010

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