Synapsis, strand scission, and strand exchange induced by the FLP recombinase: analysis with half-FRT sites.
We have used a previously described cross-linking assay and half-FRT site substrates to examine the requirements for synapsis, strand exchange, and strand scission. The cross-linking assay showed that the minimum functional FRT site needed for synapsis contains two inverted FLP-binding elements surrounding an 8-bp core. This indicates that four FLP molecules interact with four binding elements in a synaptic complex. The analysis using half-sites showed that the enzyme can catalyze efficient strand exchange between a half-site and the intact FRT site. The reaction occurred only if the half-site had at least 2 bp but no more than 4 bp of the adjoining core sequence. The exchange occurred exclusively at the regions of limited core homology between the respective half-site and the FRT site. The absence of strand exchange between an intact site and a half-site bearing regions of core nonhomology indicates that 1 bp of homology is not sufficient for the formation of stable recombinant structures. Qian et al. (X.-H. Qian, R. B. Inman, and M. M. Cox, J. Biol. Chem. 265:21779-21788, 1990) have recently shown that the FLP protein can catalyze the formation of dimeric, trimeric, and tetrameric complexes with half-FRT sites. We show that only half-sites that contained at least 2 bp of adjacent core could form stable dimer products and be cleaved by the enzyme. Stable dimers were formed between a noncleavable half-site and a cleavable half-site, suggesting that only a single cleavage event is needed for the formation of the dimer.
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Department of Medical Genetics, University of Toronto, Ontario, Canada.
Roca
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Luetke
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Sadowski
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D001482:Base Composition; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity
D004247:DNA; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase