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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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Analysis of EcoKI bound to the single-sited DNA fragments. Parts (a) and (b) show the distribution of protein volume measurements for the 1499-bp and 608-bp fragments, respectively. The complete bars represent the entire data set and the hashed areas represent proteins bound only to a specific site. Parts (c) and (d) demonstrate the relative proportions of all different protein categories in the entire analysed single-sited data sets for the 1499-bp and 608-bp fragments respectively. The abbreviated labels stand for the following: NS Mon = non-specific monomers; NS Dim = non-specific dimers; S Mon = specific monomers; S Dim = specific dimers. Note that on the looped constructs, those proteins contacting one specific site and one non-specific site were still classified as ‘specific’ (as detailed in the text).
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Figure 6: Analysis of EcoKI bound to the single-sited DNA fragments. Parts (a) and (b) show the distribution of protein volume measurements for the 1499-bp and 608-bp fragments, respectively. The complete bars represent the entire data set and the hashed areas represent proteins bound only to a specific site. Parts (c) and (d) demonstrate the relative proportions of all different protein categories in the entire analysed single-sited data sets for the 1499-bp and 608-bp fragments respectively. The abbreviated labels stand for the following: NS Mon = non-specific monomers; NS Dim = non-specific dimers; S Mon = specific monomers; S Dim = specific dimers. Note that on the looped constructs, those proteins contacting one specific site and one non-specific site were still classified as ‘specific’ (as detailed in the text).

Mentions: Detailed analysis of the size and position of EcoKI molecules on the DNA was carried out on a random selection of molecules from the 608- and 1499-bp single-sited data sets and both sets produced comparable results. As with the double-sited DNA the distribution of volume size was bimodal with one peak corresponding to EcoKI monomers and one peak corresponding to specifically bound EcoKI dimers (Figure 6a–b). For both sets each protein molecule was given one of the following classifications: non-specifically bound monomer, non-specifically bound dimer, specifically bound monomer, or specifically bound dimer. In looped complexes, most were formed by joining a specific EcoKI site to a random site and were classed as specific monomers (rare) or specific dimers (the majority). A few loops joined to random locations on the DNA and were classed as non-specific monomers or dimers. From the bar-charts using these classifications (Figure 6c–d), it was clear that a large proportion of the data was made up of non-specific monomers (67% for the long fragment and 73% for the short fragment). The remainder comprised specifically bound dimers (21% for the long fragment and 20% for the short fragment), non-specifically bound dimers (5% on long fragment and 3% on the short fragment) and specifically bound monomers (7% on the long fragment and 5% on the short fragment). The occurrence of dimerization on single-sited DNA suggests that dimerization occurs before specific sites are brought together. The relative proportions of all the data suggests that finding the initial specific site is a rate limiting factor. It also suggests that whilst dimerization is rare at non-specific sites, once specific binding occurs, dimerization is relatively straightforward. A further observation was that of the EcoKI molecules bound non-specifically, roughly 50% were bound at the end of a DNA molecule. This is as expected as the EcoKI molecule bends DNA, an energetically costly process, and this energy will not be required if the enzyme sits right on the end of a DNA molecule (33). Comparing Figure 6c and d with Figure 4f, one can see that the presence of two EcoKI sites greatly increases the proportion of EcoKI dimers bound at the EcoKI target site. The proportion of loops was also slightly greater at 16.7% presumably due to the possibility of being able to form stable loops linking the two target sites in addition to loops linking a single target site with some other random sequence. The presence of two types of loops would suggest that the formation of a loop joining two specific sites potentially arises from an intermediate with contacts to one specific and one non-specific site. Once this intermediate has been formed, the high binding affinity of EcoKI for the target sequence enhances the number of complexes with loops joining two specific target sequences.Figure 6.


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)

Analysis of EcoKI bound to the single-sited DNA fragments. Parts (a) and (b) show the distribution of protein volume measurements for the 1499-bp and 608-bp fragments, respectively. The complete bars represent the entire data set and the hashed areas represent proteins bound only to a specific site. Parts (c) and (d) demonstrate the relative proportions of all different protein categories in the entire analysed single-sited data sets for the 1499-bp and 608-bp fragments respectively. The abbreviated labels stand for the following: NS Mon = non-specific monomers; NS Dim = non-specific dimers; S Mon = specific monomers; S Dim = specific dimers. Note that on the looped constructs, those proteins contacting one specific site and one non-specific site were still classified as ‘specific’ (as detailed in the text).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: Analysis of EcoKI bound to the single-sited DNA fragments. Parts (a) and (b) show the distribution of protein volume measurements for the 1499-bp and 608-bp fragments, respectively. The complete bars represent the entire data set and the hashed areas represent proteins bound only to a specific site. Parts (c) and (d) demonstrate the relative proportions of all different protein categories in the entire analysed single-sited data sets for the 1499-bp and 608-bp fragments respectively. The abbreviated labels stand for the following: NS Mon = non-specific monomers; NS Dim = non-specific dimers; S Mon = specific monomers; S Dim = specific dimers. Note that on the looped constructs, those proteins contacting one specific site and one non-specific site were still classified as ‘specific’ (as detailed in the text).
Mentions: Detailed analysis of the size and position of EcoKI molecules on the DNA was carried out on a random selection of molecules from the 608- and 1499-bp single-sited data sets and both sets produced comparable results. As with the double-sited DNA the distribution of volume size was bimodal with one peak corresponding to EcoKI monomers and one peak corresponding to specifically bound EcoKI dimers (Figure 6a–b). For both sets each protein molecule was given one of the following classifications: non-specifically bound monomer, non-specifically bound dimer, specifically bound monomer, or specifically bound dimer. In looped complexes, most were formed by joining a specific EcoKI site to a random site and were classed as specific monomers (rare) or specific dimers (the majority). A few loops joined to random locations on the DNA and were classed as non-specific monomers or dimers. From the bar-charts using these classifications (Figure 6c–d), it was clear that a large proportion of the data was made up of non-specific monomers (67% for the long fragment and 73% for the short fragment). The remainder comprised specifically bound dimers (21% for the long fragment and 20% for the short fragment), non-specifically bound dimers (5% on long fragment and 3% on the short fragment) and specifically bound monomers (7% on the long fragment and 5% on the short fragment). The occurrence of dimerization on single-sited DNA suggests that dimerization occurs before specific sites are brought together. The relative proportions of all the data suggests that finding the initial specific site is a rate limiting factor. It also suggests that whilst dimerization is rare at non-specific sites, once specific binding occurs, dimerization is relatively straightforward. A further observation was that of the EcoKI molecules bound non-specifically, roughly 50% were bound at the end of a DNA molecule. This is as expected as the EcoKI molecule bends DNA, an energetically costly process, and this energy will not be required if the enzyme sits right on the end of a DNA molecule (33). Comparing Figure 6c and d with Figure 4f, one can see that the presence of two EcoKI sites greatly increases the proportion of EcoKI dimers bound at the EcoKI target site. The proportion of loops was also slightly greater at 16.7% presumably due to the possibility of being able to form stable loops linking the two target sites in addition to loops linking a single target site with some other random sequence. The presence of two types of loops would suggest that the formation of a loop joining two specific sites potentially arises from an intermediate with contacts to one specific and one non-specific site. Once this intermediate has been formed, the high binding affinity of EcoKI for the target sequence enhances the number of complexes with loops joining two specific target sequences.Figure 6.

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