Fusion of GFP to the M.EcoKI DNA methyltransferase produces a new probe of Type I DNA restriction and modification enzymes.
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 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.
Chen
Kai
K
School of Chemistry, University of Edinburgh, The King's Buildings, Edinburgh, EH9 3JJ, UK.
Roberts
Gareth A
GA
Stephanou
Augoustinos S
AS
Cooper
Laurie P
LP
White
John H
JH
Dryden
David T F
DT
D004247:DNA / Q000737:chemistry; D015253:Deoxyribonucleases, Type I Site-Specific / Q000737:chemistry* / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry* / Q000235:genetics
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004247:DNA; C093244:DNA modification methylase EcoKI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D015253:Deoxyribonucleases, Type I Site-Specific