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

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.

SUBMITTER: Neaves KJ

PROVIDER: S-EPMC2665228 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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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 Kelly J KJ Cooper Laurie P LP White John H JH Carnally Stewart M SM Dryden David T F DT Edwardson J Michael JM Henderson Robert M RM

Nucleic acids research 20090217 6

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 translocat ...[more]

PMID: 19223329

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Project description:Connexin proteins form hexameric assemblies known as hemichannels. When docked to form gap junction (GJ) channels, hemichannels play a critical role in cell-cell communication and cellular homeostasis, but often are functional entities on their own in unapposed cell membranes. Defects in the Connexin26 (Cx26) gene are the major cause of hereditary deafness arising from dysfunctional hemichannels in the cochlea. Structural studies of Cx26 hemichannels properly trafficked and inserted in plasma membranes, including their clustering that forms a plaque-like feature in whole gap junctions, are limited. We used atomic force microscopy (AFM) to study the surface topography of Cx26 hemichannels using two different membrane preparations. Rat Cx26 containing appended carboxy terminal V5 and hexahistidine tags were expressed in baculovirus/Sf9 cell systems. The expressed Cx26 proteins form hemichannels in situ in Sf9 cells that were then purified either as (1) Sf9 membrane fragments containing Cx26 hemichannels or (2) solubilized hemichannels. The latter were subsequently reconstituted in liposomes. AFM images of purified membrane fragments showed clusters of protein macromolecular structures in the membrane that at higher magnification corresponded to Cx26 hemichannels. Hemichannels reconstituted into DOPC bilayers displayed two populations of channel heights likely resulting from differences in orientations of inserted hemichannels. Hemichannels in the protein rich portions of purified membranes also showed a reduced channel height above the bilayer compared to membranes with reconstituted hemichannels perhaps due to reduced AFM probe access to the lipid bilayer. These preparations of purified membranes enriched for connexin hemichannels that have been properly trafficked and inserted in membranes provide a platform for high-resolution AFM imaging of the structure, interconnexon interactions, and cooperativity of properly trafficked and inserted noncrystalline connexin hemichannels.

Dataset Information

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

Publications

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

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