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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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AFM images of EcoKI on single-sited DNA. Part (a) shows a specific EcoKI dimer on the 608-bp fragment, (b) shows a non-specifically bound EcoKI monomer on the 608-bp fragment, (c) shows one specific EcoKI dimer and one non-specific monomer on the 1499-bp fragment and (d) shows a specific EcoKI dimer forming a loop on a 1499-bp fragment which also contains two non-specific EcoKI monomers.
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Figure 5: AFM images of EcoKI on single-sited DNA. Part (a) shows a specific EcoKI dimer on the 608-bp fragment, (b) shows a non-specifically bound EcoKI monomer on the 608-bp fragment, (c) shows one specific EcoKI dimer and one non-specific monomer on the 1499-bp fragment and (d) shows a specific EcoKI dimer forming a loop on a 1499-bp fragment which also contains two non-specific EcoKI monomers.

Mentions: The binding of EcoKI to the 1499-bp single-sited linear DNA fragment was carried out with a 2:1 ratio of protein to DNA in a buffer containing AdoMet and magnesium ions but in the absence of ATP to prevent translocation. 317 DNA molecules were captured from 22 AFM scans (Figure 5). Each DNA molecule contained an average of 1.2 bound protein molecules and there were 30 looped molecules with a protein at the base of the loop (9.5%). The average volume of the protein molecule at the base of each loop was 1103 nm3 (±47 nm3) consistent with each complex being formed by a protein dimer. The loops formed on single-sited DNA must be random-loops (as opposed to the double specific loops seen on the double-sited DNA). The same experiment was carried out on the 608-bp single-sited DNA fragment and negligible loop formation was observed. This was not unexpected as there are only 227 and 381 bp on either side of the target site, equivalent to ∼1.5 and ∼2.5 persistence lengths in the worm-like chain model (32,35), and the probability of loop formation will be very low.Figure 5.


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)

AFM images of EcoKI on single-sited DNA. Part (a) shows a specific EcoKI dimer on the 608-bp fragment, (b) shows a non-specifically bound EcoKI monomer on the 608-bp fragment, (c) shows one specific EcoKI dimer and one non-specific monomer on the 1499-bp fragment and (d) shows a specific EcoKI dimer forming a loop on a 1499-bp fragment which also contains two non-specific EcoKI monomers.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 5: AFM images of EcoKI on single-sited DNA. Part (a) shows a specific EcoKI dimer on the 608-bp fragment, (b) shows a non-specifically bound EcoKI monomer on the 608-bp fragment, (c) shows one specific EcoKI dimer and one non-specific monomer on the 1499-bp fragment and (d) shows a specific EcoKI dimer forming a loop on a 1499-bp fragment which also contains two non-specific EcoKI monomers.
Mentions: The binding of EcoKI to the 1499-bp single-sited linear DNA fragment was carried out with a 2:1 ratio of protein to DNA in a buffer containing AdoMet and magnesium ions but in the absence of ATP to prevent translocation. 317 DNA molecules were captured from 22 AFM scans (Figure 5). Each DNA molecule contained an average of 1.2 bound protein molecules and there were 30 looped molecules with a protein at the base of the loop (9.5%). The average volume of the protein molecule at the base of each loop was 1103 nm3 (±47 nm3) consistent with each complex being formed by a protein dimer. The loops formed on single-sited DNA must be random-loops (as opposed to the double specific loops seen on the double-sited DNA). The same experiment was carried out on the 608-bp single-sited DNA fragment and negligible loop formation was observed. This was not unexpected as there are only 227 and 381 bp on either side of the target site, equivalent to ∼1.5 and ∼2.5 persistence lengths in the worm-like chain model (32,35), and the probability of loop formation will be very low.Figure 5.

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