Structural analysis of EcoRI-DNA complexes as revealed by electron microscopy |
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Authors: | Johannssen Walther Schütte Horst Mayer Hubert Mayer Frank |
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Institution: | (1) Institut für Mikrobiologie der Universität Göttingen, Grisebachstraße 8, D-3400 Göttingen, Federal Republic of Germany;(2) Gesellschaft für Biotechnologische Forschung mbH, D-3300 Braunschweig, Federal Republic of Germany |
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Abstract: | Electron microscopy of negatively stained isolated restriction enzyme EcoRI revealed particle projections with triangular or square outlines, indicating that the enzyme, in its tetrameric state, is tetrahedron-like. The two dimers making up the tetramer appear to be arranged in two planes orthogonal to each other. Complexes formed by EcoRI with the plasmids pBR322 or pGW10 were investigated by electron microscopic spreading techniques. In the presence of Mg2+, EcoRI was bound to the DNA molecules to form pearl necklace-like aggregates. The number of bound EcoRI particles was much higher as the sum of EcoRI-and 5 ..AATT..3 sites (with exceptions, the 5 ..AATT..3 sites may function as one type of EcoRI* sites) along the DNAs, indicating unspecific binding. In the absence of Mg2+, EcoRI was bound to the DNA only at the recognition site for EcoRI and the sites where the tetranucleotide sequence 5 ..AATT..3 was present. A direct correlation of the local concentrations of the bases A and T within the flanking sequences of the binding sites with the frequency of EcoRI to the DNA was observed. Dimers and tetramers of the enzyme was found to bind to the DNA. Tetramers occasionally exhibited two binding sites for DNA as indicated by the observation of DNA loops originating at the sites of bound tetrameric EcoRI particles.Abbreviations BAC
Benzyldimethylalkylammoniumchloride
- bp
base pairs
- Kb
kilobases
- SDS
sodium dodecylsulfate
Enzymes (EC 3.1.23.13)
Restrictionendonuclease EcoRI
- (EC 3.1.23.21)
Restrictionendonuclease HindIII
- (EC 3.1.23.37)
Restrictionendonuclease SalGI
Dedicated Professor H. G. Schlegel on occasion of this 60th birthday |
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Keywords: | EcoRI EcoRI-DNA complexes EcoRI* activity Recognition sites Frequency of binding Electron microscopy |
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