Protein-based asymmetry and protein-protein interactions in FLP recombinase-mediated site-specific recombination.
When the FLP recombination target (FRT) is cut in half so that only one FLP protein-binding site is present, FLP protein forms a complex in which two such sites are linked head to head. Although held together exclusively by noncovalent interactions, this complex survives electrophoresis in an agarose gel and exhibits a half-life that can be measured in hours. Characterization of this complex indicates that a very stable, asymmetric dimeric complex of FLP protein monomers bound to the FRT is a likely early intermediate in FLP-mediated site-specific recombination. The apparent asymmetry is a property of the protein components of the complex. Even though the DNA components form a perfect palindrome, only one of the two possible DNA cleavage steps takes place in the course of complex formation. Formation of this complex does not occur with half-FRT site DNA substrates that preclude head to head monomer contact or when a FLP mutant protein is used that binds the FRT site but cannot cleave it. Trimeric and tetrameric complexes are also observed, the latter at very low frequency. These results are discussed in terms of an expanded model for early events in FLP-mediated site-specific recombination.
Qian
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XH
Program in Cell and Molecular Biology, College of Agricultural and Life Sciences, University of Wisconsin, Madison 53706.
Inman
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RB
Cox
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D001483:Base Sequence; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D011485:Protein Binding; D011995:Recombination, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D046911:Macromolecular Substances; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase