Geometry of site alignment during int family recombination: antiparallel synapsis by the Flp recombinase.
The Flp site-specific recombinase functions in the copy number amplification of the yeast 2 microm plasmid. The recombination reaction is catalyzed by four monomers of Flp bound to two separate, but identical, recombination sites (FRT sites) and occurs in two sequential pairs of strand exchanges. The relative orientation of the two recombination sites during synapsis was examined. Topoisomerase relaxation and nick ligation were used to detect topological nodes introduced by the synapse prior to the chemical steps of recombination. A single negative supercoil was found to be trapped by Flp in substrates with inverted FRT sites whereas no trapped supercoils were observed with direct repeats. The topology of products resulting from Flp-mediated recombination adjacent to a well characterised synapse, that of Tn3 resolvase/res, was analyzed. The deletion and inversion reactions yielded the four noded catenane and the three noded knot, respectively, as the simplest and the most abundant products. The linking number change introduced by the Flp-mediated inversion reaction was determined to be +/-2. The most parsimonious explanation of these results is that Flp aligns its recombination sites with antiparallel geometry. The majority of synapses appear to occur without entrapment of additional random plectonemic DNA supercoils between the sites and no additional crossings are introduced as a result of the chemical steps of recombination.
Grainge
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Section of Molecular Genetics and Microbiology and The Institute for Cell and Molecular Biology, Austin, TX 78712, USA. grainge@mail.utexas.edu
Buck
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Jayaram
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D001665:Binding Sites; D007446:Chromosome Inversion; D003434:Crossing Over, Genetic / Q000235:genetics; D011088:DNA Ligases / Q000378:metabolism; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000378:metabolism; D004278:DNA, Superhelical / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D006801:Humans; D019426:Integrases / Q000378:metabolism*; D008957:Models, Genetic; D005810:Multigene Family; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D045522:Recombinases; D011995:Recombination, Genetic / Q000235:genetics*; D012440:Saccharomyces / Q000201:enzymology* / Q000235:genetics*; D017384:Sequence Deletion / Q000235:genetics; D013379:Substrate Specificity; D019895:Transposases / Q000378:metabolism; D045564:Transposon Resolvases; D014764:Viral Proteins
D004278:DNA, Superhelical; D045522:Recombinases; D014764:Viral Proteins; C045073:Cre recombinase; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D019426:Integrases; C473491:Tn3 resolvase; D019895:Transposases; D045564:Transposon Resolvases; D003850:Deoxyribonuclease I; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II; D011088:DNA Ligases