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Directed protein replacement in recombination full sites reveals trans‐horizontal DNA cleavage by Flp recombinase.
Author(s) -
Lee J.,
Whang I.,
Lee J.,
Jayaram M.
Publication year - 1994
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1002/j.1460-2075.1994.tb06869.x
Subject(s) - biology , recombinase , flp frt recombination , site specific recombination , recombination , genetics , dna , cleavage (geology) , gene , genetic recombination , paleontology , fracture (geology)
One round of site‐specific recombination between two DNA partners mediated by the Flp recombinase requires the breakage and reformation of four phosphodiester bonds. The reaction is accomplished by the combined action of four Flp monomers. Within the recombination complex, what is the relative disposition of a Flp monomer with respect to the target diester that it cleaves? To address this question, we have devised a strategy for the targeted orientation of Flp monomers within full‐site recombination substrates. Our experimental design is not dependent on ‘altered binding specificity’ of the recombinase. Analysis of the pattern of DNA cleavage by this method reveals no evidence for DNA cleavage in cis. A Flp monomer bound to its recognition element within the full site does not cleave the scissile phosphodiester bond adjacent to it. Our results are most consistent with ‘trans‐horizontal cleavage’. Cleavage by Flp occurs at the scissile phosphodiester distal to it, but within the same full site. The general experimental design employed here will be of widespread utility in mechanistic analyses of nucleic acid transactions involving multimeric DNA‐protein assemblies.