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 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.
Highlights
Since their introduction into genetic engineering, the green fluorescent protein (GFP) and its many spectral variants have proved to be extraordinarily useful probes of protein structure and function both in vitro and in vivo [1]
Förster resonance energy transfer (FRET) to measure distances between two fluorophores, a donor and an acceptor, has been the subject of many uses of GFP despite its complex photophysics and its relatively large size compared to more traditional small molecule fluorophores such as fluorescein [2]
In this paper we demonstrate the preparation of an active M.EcoKI fused to GFP and measure via FRET the distance from the GFP to a HEX label on a duplex bound to the MTase and to a fluorescently-labelled ocr protein bound to the MTase
Summary
Since their introduction into genetic engineering, the green fluorescent protein (GFP) and its many spectral variants have proved to be extraordinarily useful probes of protein structure and function both in vitro and in vivo [1]. Sequence-specific DNA-binding enzymes such as methyltransferases (MTases) and endonucleases comprising bacterial restriction–modification (R/M) systems would seem to present excellent targets for analysis via fusion to GFP given that many of them introduce complex rearrangements of DNA structure including for example DNA looping to bring distant sites on a single DNA molecule into close proximity. R/M systems typically comprise a REase that recognises a specific nucleotide sequence prior to cleavage, and a cognate DNA MTase able, by methylating adenine or cytosine within the same sequence, to confer protection from the REase.
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