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: Recently Kennaway et al.  have published an atomic model of the M.EcoKI MTase bound to a DNA duplex and to ocr. Fig. 3 shows the HsdS subunit bound to DNA with the GFP chromophore placed roughly at the distances determined by FRET using the average fluorescence lifetimes (the ocr protein roughly takes the place of the DNA in the atomic model of M.EcoKI and ocr). It can be seen that the results all converge on approximately the same location for the GFP apart from the distance to the S68C location on the ocr protein. The GFP is best located directly below one end of the HsdS subunit to satisfy the FRET distances. This location is what would be expected from the model of M.EcoKI MTase as the location of the C-terminus of HsdS.
Affiliation: School of Chemistry, University of Edinburgh, The King's Buildings, Edinburgh, EH9 3JJ, UK.