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

Chen K, Roberts GA, Stephanou AS, Cooper LP, White JH, Dryden DT - Biochem. Biophys. Res. Commun. (2010)

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.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry, University of Edinburgh, The King's Buildings, Edinburgh, EH9 3JJ, UK.

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Spectrophotometric analyses. (A) Absorption spectra of 1 μM GFP-MTase (bold solid line), 1 μM 21TH21B DNA (thin solid line) and 5 μM Dylight549-labeled ocr E20C mutant protein (dashed line). Other labeled proteins had similar spectra. (B) Emission spectra of 1 μM GFP-MTase (bold solid line, excitation at 395 nm), 400 nM 21TH21B DNA (thin solid line, excitation at 530 nm) and 1 μM Dylight549-labeled ocr E20C mutant protein (dashed line, excitation at 550 nm). Other labeled proteins had similar spectra.
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fig1: Spectrophotometric analyses. (A) Absorption spectra of 1 μM GFP-MTase (bold solid line), 1 μM 21TH21B DNA (thin solid line) and 5 μM Dylight549-labeled ocr E20C mutant protein (dashed line). Other labeled proteins had similar spectra. (B) Emission spectra of 1 μM GFP-MTase (bold solid line, excitation at 395 nm), 400 nM 21TH21B DNA (thin solid line, excitation at 530 nm) and 1 μM Dylight549-labeled ocr E20C mutant protein (dashed line, excitation at 550 nm). Other labeled proteins had similar spectra.

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.


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

Chen K, Roberts GA, Stephanou AS, Cooper LP, White JH, Dryden DT - Biochem. Biophys. Res. Commun. (2010)

Spectrophotometric analyses. (A) Absorption spectra of 1 μM GFP-MTase (bold solid line), 1 μM 21TH21B DNA (thin solid line) and 5 μM Dylight549-labeled ocr E20C mutant protein (dashed line). Other labeled proteins had similar spectra. (B) Emission spectra of 1 μM GFP-MTase (bold solid line, excitation at 395 nm), 400 nM 21TH21B DNA (thin solid line, excitation at 530 nm) and 1 μM Dylight549-labeled ocr E20C mutant protein (dashed line, excitation at 550 nm). Other labeled proteins had similar spectra.
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Related In: Results  -  Collection

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fig1: Spectrophotometric analyses. (A) Absorption spectra of 1 μM GFP-MTase (bold solid line), 1 μM 21TH21B DNA (thin solid line) and 5 μM Dylight549-labeled ocr E20C mutant protein (dashed line). Other labeled proteins had similar spectra. (B) Emission spectra of 1 μM GFP-MTase (bold solid line, excitation at 395 nm), 400 nM 21TH21B DNA (thin solid line, excitation at 530 nm) and 1 μM Dylight549-labeled ocr E20C mutant protein (dashed line, excitation at 550 nm). Other labeled proteins had similar spectra.
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.

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.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry, University of Edinburgh, The King's Buildings, Edinburgh, EH9 3JJ, UK.

Show MeSH