Interactions between distantly separated DNA regions mediated by specialized proteins lead to the formation of synaptic protein-DNA complexes. This is a ubiquitous phenomenon which is critical in various genetic processes. Although such interactions typically occur between two sites, interactions among three specific DNA regions have been identified, and a corresponding model has been proposed. Atomic force microscopy was used to test this model for the EcoRII restriction enzyme and provide

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... ct visualization and characterization of synaptic protein-DNA complexes involving three DNA binding sites. The complex appeared in the images as a two-loop structure, and the length measurements proved the site specificity of the protein in the complex. The protein volume measurements showed that an EcoRII dimer is the core of the three-site synaptosome. Other complexes were identified and analyzed. The protein volume data showed that the dimeric form of the protein is responsible for the formation of other types of synaptic complexes as well. The applications of these results to the mechanisms of the protein-DNA interactions are discussed. Synaptic DNA-protein complexes (synaptosomes) are formed by DNA helices brought together and stabilized by a specialized protein or protein complexes. The formation of synaptic DNA-protein complexes is the key step of various genetic processes such as site-specific recombination, genome integration, excision, and inversion of specific DNA regions (1). In fact, the formation of a synaptic complex is a more general phenomenon that is not limited to site-specific recombination, insertion, and integration systems. There is a family of DNA restriction enzymes that require the formation of synaptic complexes for further site-specific DNA cleavage (2-5). In the majority of synaptic protein-DNA complexes, two DNA duplexes are involved in the synaptosome assembly. However, there are a number of reports indicating that more than two specific DNA regions can be involved in the formation of synaptic complexes. For example, a three-site synaptic complex is formed during Mu DNA transposition (6, 7); in the His-mediated inversion reaction, a tripartite complex is formed when the His tetramer, holding hixL and hixR sites, interacts with the Fis dimer, which brings an enhancer site to the complex (8-12). These are complex multiprotein systems in which participation of multimers of the same protein (three MuA transposases for the Mu system) or more than two proteins (His-mediated invertasome) are involved. Very recent data from the Siksnys lab showed that efficient cleavage mediated by EcoRII restriction endonuclease also requires the formation of a synaptic complex involving three DNA recognition regions (13). EcoRII is a type IIE endonuclease, which cuts duplex DNA containing the sequence /CCWGG, with W being adenine (A) or thymine (T) (cleavage position indicated by the slash). Generally, EcoRII exists in solution as a dimer (∼90 kDa) that is capable of forming synaptic complexes via cooperative binding of two DNA regions (14). According to crystallographic data of EcoRII mutant R88A (15), the protein dimer is formed through the formation of helix bundles at the dimer interface, and both N-and C-domains participate in the dimerization. The model (13) suggests that the dimeric form of the enzyme is involved in the three-site synaptosome formation. To concertedly cleave the DNA strand, three sites need to be involved in the reaction, which includes the catalytic C-terminal domain and the two N-terminal domains. Exogenic oligonucleotides added to the reaction mixture increased the reaction velocity and changed the reaction pattern for EcoRII cleavage of one-or two-site plasmids but had little effect on the three-site plasmid (13). To test this novel model for synaptic complex formation, we utilized AFM 1 to directly visualize the complexes formed by the EcoRII enzyme and DNA templates with a well-defined number and positioning of protein binding sites. EcoRII complexes have been previously visualized with electron microscopy, but formation of a three-site complex has not been reported (16). AFM has been successfully applied to various protein-DNA complexes (e.g., refs 17-26 and