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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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The HsdS subunit bound to a DNA duplex as proposed from electron microscopy data [7] is shown above a GFP model with the chromophore shown in the centre of the GFP β-barrel. The locations of the HEX labels (21TH21B is on the left and 21T21BH is on the right) and of the locations of the ocr residues labelled with Dylight549 are indicated (ocr is not shown but superimposes on and extends further out than the DNA duplex shown). The arrows show the FRET distances determined from <τ> given in Table 3 except for the distance to S68 on ocr where the distance in the actual model is shown (the FRET distance is longer for this pair but is incorrect due to rotational constraints on the acceptor).
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fig3: The HsdS subunit bound to a DNA duplex as proposed from electron microscopy data [7] is shown above a GFP model with the chromophore shown in the centre of the GFP β-barrel. The locations of the HEX labels (21TH21B is on the left and 21T21BH is on the right) and of the locations of the ocr residues labelled with Dylight549 are indicated (ocr is not shown but superimposes on and extends further out than the DNA duplex shown). The arrows show the FRET distances determined from <τ> given in Table 3 except for the distance to S68 on ocr where the distance in the actual model is shown (the FRET distance is longer for this pair but is incorrect due to rotational constraints on the acceptor).

Mentions: Recently Kennaway et al. [7] 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.


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)

The HsdS subunit bound to a DNA duplex as proposed from electron microscopy data [7] is shown above a GFP model with the chromophore shown in the centre of the GFP β-barrel. The locations of the HEX labels (21TH21B is on the left and 21T21BH is on the right) and of the locations of the ocr residues labelled with Dylight549 are indicated (ocr is not shown but superimposes on and extends further out than the DNA duplex shown). The arrows show the FRET distances determined from <τ> given in Table 3 except for the distance to S68 on ocr where the distance in the actual model is shown (the FRET distance is longer for this pair but is incorrect due to rotational constraints on the acceptor).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2914225&req=5

fig3: The HsdS subunit bound to a DNA duplex as proposed from electron microscopy data [7] is shown above a GFP model with the chromophore shown in the centre of the GFP β-barrel. The locations of the HEX labels (21TH21B is on the left and 21T21BH is on the right) and of the locations of the ocr residues labelled with Dylight549 are indicated (ocr is not shown but superimposes on and extends further out than the DNA duplex shown). The arrows show the FRET distances determined from <τ> given in Table 3 except for the distance to S68 on ocr where the distance in the actual model is shown (the FRET distance is longer for this pair but is incorrect due to rotational constraints on the acceptor).
Mentions: Recently Kennaway et al. [7] 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.

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