Fusion of GFP to the M.EcoKI DNA methyltransferase produces a new probe of Type I DNA restriction and modification enzymes.
Bottom Line: The fusion protein functions as a sequence-specific DNA methyltransferase protecting DNA against digestion by the EcoKI restriction endonuclease.The purified enzyme shows Förster 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.
Affiliation: School of Chemistry, University of Edinburgh, The King's Buildings, Edinburgh, EH9 3JJ, UK.Show MeSH
Mentions: The purified GFP-MTase showed the absorption and fluorescence emission properties expected, Fig. 1A, B. The overlap of the emission of the GFP with the HEX and Dylight549 labels allowed Ro distances of 6.14 nm and 6.53 nm, respectively, to be calculated.
Affiliation: School of Chemistry, University of Edinburgh, The King's Buildings, Edinburgh, EH9 3JJ, UK.