Reactions between half- and full-FLP recombination target sites. A model system for analyzing early steps in FLP protein-mediated site-specific recombination.
The FLP recombination target (FRT) can be cut in half so that only one FLP protein binding site is present (a "half site"). FLP protein binds the half sites and joins them into dimeric, asymmetric head-to-head complexes held together chiefly by strong noncovalent interactions. These complexes react with full (normal) FRT sites to generate a variety of products. Analysis of these DNA species reveals that the reaction follows a well-defined reaction pathway that generally parallels the normal reaction pathway. The system is useful in analyzing early steps in recombination, since the identity of the products in a given recombination event unambiguously pinpoints the order in which the cleavage and strand exchange reactions occur. Two conclusions are derived from the present study: (i) Formation of the dimeric head-to-head complex of half sites is a prerequisite to further steps in recombination. (ii) The identity of the base pairs at positions 6 and -6 within the FRT site has a subtle effect in directing the first strand exchange event in the reaction to predominantly one of two possible cleavage sites. In addition, results are presented that suggest that a DNA-DNA pairing intermediate involving only two base pairs of the core sequence is formed prior to the first cleavage and strand exchange. DNA-DNA interactions may therefore not be limited to the isomerization step that follows the first strand exchange.
Qian
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XH
Department of Biochemistry, 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; D004247:DNA / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D008854:Microscopy, Electron; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010957:Plasmids; D011995:Recombination, Genetic; D013379:Substrate Specificity
D004247:DNA; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase