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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 representations of EcoKI binding, translocation and restriction on DNA. Part (a) shows the previous model. In this model EcoKI monomers individually bind to each site and translocation occurs independently from both occupied sites. When the monomers meet, translocation is stalled and restriction occurs. Part (b) shows a new model, which is based on the existing model but incorporates the additional information discovered in this paper and in (8,23). In this new model a monomer of EcoKI binds to one site and then a second monomer binds to the same site to form a dimerized complex at that site. This dimerized complex then forms diffusive loops with non-specific regions of DNA until it is stabilized by contact with the secondary EcoKI site. Translocation then occurs from both sides of both monomers and, in agreement with the previous model, restriction occurs when the translocation process is stalled (this time because the diffusive loop between the monomers becomes fully contracted). In the evidence presented here it remains unanswered whether translocation is triggered by the occupation of a secondary site or whether both processes occur concurrently. In both models DNA is represented as a line, specific EcoKI sites are represented by dots on the DNA molecules, and EcoKI monomers are represented as individual spherical objects.
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Figure 7: Diagrammatic representations of EcoKI binding, translocation and restriction on DNA. Part (a) shows the previous model. In this model EcoKI monomers individually bind to each site and translocation occurs independently from both occupied sites. When the monomers meet, translocation is stalled and restriction occurs. Part (b) shows a new model, which is based on the existing model but incorporates the additional information discovered in this paper and in (8,23). In this new model a monomer of EcoKI binds to one site and then a second monomer binds to the same site to form a dimerized complex at that site. This dimerized complex then forms diffusive loops with non-specific regions of DNA until it is stabilized by contact with the secondary EcoKI site. Translocation then occurs from both sides of both monomers and, in agreement with the previous model, restriction occurs when the translocation process is stalled (this time because the diffusive loop between the monomers becomes fully contracted). In the evidence presented here it remains unanswered whether translocation is triggered by the occupation of a secondary site or whether both processes occur concurrently. In both models DNA is represented as a line, specific EcoKI sites are represented by dots on the DNA molecules, and EcoKI monomers are represented as individual spherical objects.

Mentions: Our results suggest the following mechanism for EcoKI. First, an EcoKI monomer binds to DNA at a random sequence and then, in common with other DNA-binding proteins, it may either dissociate or conduct a limited degree of linear diffusion up and down the DNA contour (35). During this diffusion process the enzyme may find its target sequence. A second monomer can bind to the EcoKI already on the DNA to form a dimer and this dimer is more stable if it forms at a target sequence. A dimer bound at the target site then collides with another region of DNA due to the inherent flexibility of the DNA to form a loop. This second location on the DNA can be either a second specificity site or, more probably, a non-specific DNA region. Once the loop is formed, the second EcoKI molecule can scan the DNA contour for another copy of the target sequence. The loop may fall apart during this process so multiple rounds of loop formation may be required before the second target sequence is found. Despite the possibility of repetitive rounds of loop formation, the process is still an efficient means for locating the second site (35). Once both target sequences have been located by the dimer of EcoKI, ATPase-driven translocation and DNA cleavage can be initiated. This dimerization of EcoKI prior to translocation can be incorporated into the existing model for EcoKI translocation and restriction, as shown in Figure 7.Figure 7.


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 representations of EcoKI binding, translocation and restriction on DNA. Part (a) shows the previous model. In this model EcoKI monomers individually bind to each site and translocation occurs independently from both occupied sites. When the monomers meet, translocation is stalled and restriction occurs. Part (b) shows a new model, which is based on the existing model but incorporates the additional information discovered in this paper and in (8,23). In this new model a monomer of EcoKI binds to one site and then a second monomer binds to the same site to form a dimerized complex at that site. This dimerized complex then forms diffusive loops with non-specific regions of DNA until it is stabilized by contact with the secondary EcoKI site. Translocation then occurs from both sides of both monomers and, in agreement with the previous model, restriction occurs when the translocation process is stalled (this time because the diffusive loop between the monomers becomes fully contracted). In the evidence presented here it remains unanswered whether translocation is triggered by the occupation of a secondary site or whether both processes occur concurrently. In both models DNA is represented as a line, specific EcoKI sites are represented by dots on the DNA molecules, and EcoKI monomers are represented as individual spherical objects.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 7: Diagrammatic representations of EcoKI binding, translocation and restriction on DNA. Part (a) shows the previous model. In this model EcoKI monomers individually bind to each site and translocation occurs independently from both occupied sites. When the monomers meet, translocation is stalled and restriction occurs. Part (b) shows a new model, which is based on the existing model but incorporates the additional information discovered in this paper and in (8,23). In this new model a monomer of EcoKI binds to one site and then a second monomer binds to the same site to form a dimerized complex at that site. This dimerized complex then forms diffusive loops with non-specific regions of DNA until it is stabilized by contact with the secondary EcoKI site. Translocation then occurs from both sides of both monomers and, in agreement with the previous model, restriction occurs when the translocation process is stalled (this time because the diffusive loop between the monomers becomes fully contracted). In the evidence presented here it remains unanswered whether translocation is triggered by the occupation of a secondary site or whether both processes occur concurrently. In both models DNA is represented as a line, specific EcoKI sites are represented by dots on the DNA molecules, and EcoKI monomers are represented as individual spherical objects.
Mentions: Our results suggest the following mechanism for EcoKI. First, an EcoKI monomer binds to DNA at a random sequence and then, in common with other DNA-binding proteins, it may either dissociate or conduct a limited degree of linear diffusion up and down the DNA contour (35). During this diffusion process the enzyme may find its target sequence. A second monomer can bind to the EcoKI already on the DNA to form a dimer and this dimer is more stable if it forms at a target sequence. A dimer bound at the target site then collides with another region of DNA due to the inherent flexibility of the DNA to form a loop. This second location on the DNA can be either a second specificity site or, more probably, a non-specific DNA region. Once the loop is formed, the second EcoKI molecule can scan the DNA contour for another copy of the target sequence. The loop may fall apart during this process so multiple rounds of loop formation may be required before the second target sequence is found. Despite the possibility of repetitive rounds of loop formation, the process is still an efficient means for locating the second site (35). Once both target sequences have been located by the dimer of EcoKI, ATPase-driven translocation and DNA cleavage can be initiated. This dimerization of EcoKI prior to translocation can be incorporated into the existing model for EcoKI translocation and restriction, as shown in Figure 7.Figure 7.

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
Related in: MedlinePlus