Bending-incompetent variants of Flp recombinase mediate strand transfer in half-site recombinations: role of DNA bending in recombination.
One key feature of the interaction of Flp recombinase with its target site (FRT) is the large bend introduced in the substrate as a result of protein binding. The extent of bending was found to depend on the phasing and spacing of the Flp monomers occupying the two Flp-binding elements (FBE) bordering the strand-exchange region (spacer) of the substrate. The relative mobilities of the Flp complexes formed by the two permuted substrate fragments, containing the FRT site near the end or in the middle, corresponded to a DNA bend of approx. 140 degrees when each of the two FBEs flanking the spacer was occupied by a protein monomer. The estimated bend angle was the same when the reference DNA fragment with the FRT site at the end was substituted by one with the site in the middle, but containing a 4-bp insertion within the spacer. We used a combination of wild-type Flp and Flp variants that were competent or incompetent in DNA bending, together with full, or half FRT sites, to ask whether bending is a conformational requirement for catalysis, namely cleavage and exchange of strands. We obtained the following results: in full-site (FRT) vs. full-site recombinations or in full-site vs. half-site (half FRT) recombinations, there was a large difference in the reactivity between Flp and a bending-incompetent Flp variant. This difference virtually disappeared when reactions were done with half-FRT sites. We conclude that bending is not a prerequisite for catalysis, but represents the manner in which the substrate accommodates the Flp protomer-protomer interactions that are pertinent to catalysis.
Chen
J W
JW
Department of Microbiology, University of Texas, Austin 78712.
Evans
B
B
Rosenfeldt
H
H
Jayaram
M
M
D001483:Base Sequence; D002384:Catalysis; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D004271:DNA, Fungal / Q000737:chemistry / Q000378:metabolism*; D014644:Genetic Variation; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D014443:Tyrosine / Q000235:genetics
D004271:DNA, Fungal; D014443:Tyrosine; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase