Abstract
To determine the physiologically important features of site-specific recombination by Escherichia coli Tn3 resolvase we analyzed the salient properties of the reaction in vivo. A two-plasmid system in which one plasmid served as substrate while the other encoded both resolvase and a thermolabile repressor of resolvase transcription provided controlled, synchronous recombination. Recombination proceeded rapidly and was promoted by (-) DNA supercoiling. The structures of the in vivo recombination products were predominantly the same as those previously identified in vitro. By examination of the products of successive rounds of recombination of a four-site substrate, we ruled out a tracking mechanism for site alignment. Inversion and plasmid fusion occurred in vivo at a much lower rate than resolution but ultimately reached a higher extent than found in vitro. We propose that inversion and fusion exploit topologically interlinked substrates that occur at low levels in vivo. This proposal is supported by the unexpected specificity of fusion. Our data imply that supercoiled DNA, the resolvase synaptic complex, and the mechanism of strand exchange are fundamentally similar in vivo and in vitro, but that the repertoire of resolvase substrates and products is expanded in vivo by the action of other enzymes that alter DNA topology.
Highlights
It is important to establish the propeorftireesolvase action in uiuo, as i n uitro studies show thattherequirement for than resolution but reached a higher extent negative supercoilingand the unidirectionalitoyf the reaction than found in vitro
The reactionoccurs only if the res sites are directly repeated in the same molecule, just as they arepositioned in a natural cointegrate
From these observations and from the structure of products from multiple rounds of recombination, we conclude that themechanism of site synapsis and strand exchange are the same in viuo as established in uitro
Summary
The resolvase of the recombinants formed before gyrase was fully inhibited, recombination products generated in uitro from this substrate because 35% of the substrate was recombined at thetime of were nicked by DNase I and displayed by high resolution gel norfloxacin addition (lane 2). Additionof norfloxacin at 1.5 min proved optimal for blocking decatenation while still allowing multiple rounds of recombination Under these conditions, the products of the second and third rounds (Fig. 4, lune2 ) had the sameelectrophoreticmobilities as theproducts formed inuitro (Fig. 4, lane 1). The in vitro of nicked pA4 recombinatiopnroducts duplicate to that shown first round product distributions calculated by this method agree to, lane 2, was excised and the DNA was digested in within 1%of the data previously determined by gel electrophoresis situ withEcoRI. From the position of the two possible first round products (pA3.A and pA2.A2),the
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