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Atomic force microscopy of the EcoKI Type I DNA restriction enzyme bound to DNA shows enzyme dimerization and DNA looping.

Neaves KJ, Cooper LP, White JH, Carnally SM, Dryden DT, Edwardson JM, Henderson RM - Nucleic Acids Res. (2009)

Bottom Line: The results presented here extend earlier findings confirming the dimerization.Visualization of specific DNA loops in the protein-DNA constructs was achieved by improved sample preparation and analysis techniques.The reported dimerization and looping mechanism is unlikely to be exclusive to EcoKI, and offers greater insight into the detailed functioning of this and other higher order assemblies of proteins operating by bringing distant sites on DNA into close proximity via DNA looping.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Cambridge, Cambridge, UK.

ABSTRACT
Atomic force microscopy (AFM) allows the study of single protein-DNA interactions such as those observed with the Type I Restriction-Modification systems. The mechanisms employed by these systems are complicated and understanding them has proved problematic. It has been known for years that these enzymes translocate DNA during the restriction reaction, but more recent AFM work suggested that the archetypal EcoKI protein went through an additional dimerization stage before the onset of translocation. The results presented here extend earlier findings confirming the dimerization. Dimerization is particularly common if the DNA molecule contains two EcoKI recognition sites. DNA loops with dimers at their apex form if the DNA is sufficiently long, and also form in the presence of ATPgammaS, a non-hydrolysable analogue of the ATP required for translocation, indicating that the looping is on the reaction pathway of the enzyme. Visualization of specific DNA loops in the protein-DNA constructs was achieved by improved sample preparation and analysis techniques. The reported dimerization and looping mechanism is unlikely to be exclusive to EcoKI, and offers greater insight into the detailed functioning of this and other higher order assemblies of proteins operating by bringing distant sites on DNA into close proximity via DNA looping.

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Diagrammatic representation of (a) the 1499-bp double-sited DNA substrate (b) the 1499-bp single-sited DNA substrate (c) the 1499-bp zero-sited fragment and (d) the 608-bp single-sited fragment. (a) and (d) were constructed from the mutated pBRsK15 and (b) and (c) were constructed from pBRsK15. The restriction enzymes used to produce the individual fragments are written at the end of each fragment. In each diagram the DNA is represented by a line and EcoKI-binding sites are represented by spheres. The centre of the each sphere, and the figure above each sphere, corresponds to the central base pair in the EcoKI-binding sequence. The numbers below each DNA fragment show the distance between binding sites and/or the distance from each binding site to the end of the DNA molecule. The width of each sphere is a proportional representation of the DNA footprint of EcoKI. However, in AFM imaging the widths would appear to be much greater due to tip convolution.
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Figure 2: Diagrammatic representation of (a) the 1499-bp double-sited DNA substrate (b) the 1499-bp single-sited DNA substrate (c) the 1499-bp zero-sited fragment and (d) the 608-bp single-sited fragment. (a) and (d) were constructed from the mutated pBRsK15 and (b) and (c) were constructed from pBRsK15. The restriction enzymes used to produce the individual fragments are written at the end of each fragment. In each diagram the DNA is represented by a line and EcoKI-binding sites are represented by spheres. The centre of the each sphere, and the figure above each sphere, corresponds to the central base pair in the EcoKI-binding sequence. The numbers below each DNA fragment show the distance between binding sites and/or the distance from each binding site to the end of the DNA molecule. The width of each sphere is a proportional representation of the DNA footprint of EcoKI. However, in AFM imaging the widths would appear to be much greater due to tip convolution.

Mentions: Two separate linear products were produced using the mutated pBRsK15 plasmid and a further two products were created from the original non-mutated pBRsK15 plasmid. Figure 2 shows a diagrammatic representation of all the linear DNA products.Figure 2.


Atomic force microscopy of the EcoKI Type I DNA restriction enzyme bound to DNA shows enzyme dimerization and DNA looping.

Neaves KJ, Cooper LP, White JH, Carnally SM, Dryden DT, Edwardson JM, Henderson RM - Nucleic Acids Res. (2009)

Diagrammatic representation of (a) the 1499-bp double-sited DNA substrate (b) the 1499-bp single-sited DNA substrate (c) the 1499-bp zero-sited fragment and (d) the 608-bp single-sited fragment. (a) and (d) were constructed from the mutated pBRsK15 and (b) and (c) were constructed from pBRsK15. The restriction enzymes used to produce the individual fragments are written at the end of each fragment. In each diagram the DNA is represented by a line and EcoKI-binding sites are represented by spheres. The centre of the each sphere, and the figure above each sphere, corresponds to the central base pair in the EcoKI-binding sequence. The numbers below each DNA fragment show the distance between binding sites and/or the distance from each binding site to the end of the DNA molecule. The width of each sphere is a proportional representation of the DNA footprint of EcoKI. However, in AFM imaging the widths would appear to be much greater due to tip convolution.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2665228&req=5

Figure 2: Diagrammatic representation of (a) the 1499-bp double-sited DNA substrate (b) the 1499-bp single-sited DNA substrate (c) the 1499-bp zero-sited fragment and (d) the 608-bp single-sited fragment. (a) and (d) were constructed from the mutated pBRsK15 and (b) and (c) were constructed from pBRsK15. The restriction enzymes used to produce the individual fragments are written at the end of each fragment. In each diagram the DNA is represented by a line and EcoKI-binding sites are represented by spheres. The centre of the each sphere, and the figure above each sphere, corresponds to the central base pair in the EcoKI-binding sequence. The numbers below each DNA fragment show the distance between binding sites and/or the distance from each binding site to the end of the DNA molecule. The width of each sphere is a proportional representation of the DNA footprint of EcoKI. However, in AFM imaging the widths would appear to be much greater due to tip convolution.
Mentions: Two separate linear products were produced using the mutated pBRsK15 plasmid and a further two products were created from the original non-mutated pBRsK15 plasmid. Figure 2 shows a diagrammatic representation of all the linear DNA products.Figure 2.

Bottom Line: The results presented here extend earlier findings confirming the dimerization.Visualization of specific DNA loops in the protein-DNA constructs was achieved by improved sample preparation and analysis techniques.The reported dimerization and looping mechanism is unlikely to be exclusive to EcoKI, and offers greater insight into the detailed functioning of this and other higher order assemblies of proteins operating by bringing distant sites on DNA into close proximity via DNA looping.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Cambridge, Cambridge, UK.

ABSTRACT
Atomic force microscopy (AFM) allows the study of single protein-DNA interactions such as those observed with the Type I Restriction-Modification systems. The mechanisms employed by these systems are complicated and understanding them has proved problematic. It has been known for years that these enzymes translocate DNA during the restriction reaction, but more recent AFM work suggested that the archetypal EcoKI protein went through an additional dimerization stage before the onset of translocation. The results presented here extend earlier findings confirming the dimerization. Dimerization is particularly common if the DNA molecule contains two EcoKI recognition sites. DNA loops with dimers at their apex form if the DNA is sufficiently long, and also form in the presence of ATPgammaS, a non-hydrolysable analogue of the ATP required for translocation, indicating that the looping is on the reaction pathway of the enzyme. Visualization of specific DNA loops in the protein-DNA constructs was achieved by improved sample preparation and analysis techniques. The reported dimerization and looping mechanism is unlikely to be exclusive to EcoKI, and offers greater insight into the detailed functioning of this and other higher order assemblies of proteins operating by bringing distant sites on DNA into close proximity via DNA looping.

Show MeSH