The shuffling function of resolvases.

Redistribution (shuffling) of genetic material between replicons requires consecutive recombination at four points, two of which (X, X') are involved in the replicon fusion and the other two (Y, Y'), in the cointegrate resolution. The fusion of replicons by bacterial resolvases makes the second recombination round at sites Y and Y' problematic because of the high probability of the reverse reaction. Structural differences of the res sites recognized by resolvase could delay the reverse reaction, thus enhancing the probability of recombination at sites Y, Y', but the direct reaction ensuring it (i.e. the fusion of replicons via different res sites) has not been described yet. Here, a genetic system to test intermolecular recombination at heterogeneous res sites has been developed. The system was based on the res site (RS2) of the novel resolution system, cinH-RS2, encoded by pKLH2, pKLH204 and pKLH205. As its partner, the res site of RP4 located in the par locus, the res site of transposon gammadelta or Tn1721, the incomplete site RS1 consisting only of the (crossover) subsite resI also found in pKLH2/204/205 and others were used. Except for the pairing of RS2 x gammadelta res, recombination was observed in each case even when the homology shared by partners did not exceed 35% (as in RS2 x par). In the latter case, the presence in cis of an additional, enhancer-like-acting element was required. Pairing of crossover subsites during site-specific recombination occurred in either orientation, depending on the structure of res partners and the kind of resolvase acting on the sites. With the complete res sites, the antiparallel alignment resulted in the production of an unusual res having the accessory subsites II and III at both sides of I, and a res lacking II and III. The wide range of frequencies was observed not only in the fusion formation but also in the dissociation of the resulting cointegrates. Hence, the resolvase-mediated interreplicon exchange of the DNA segments by fusion via an inefficient reaction (at sites X, X') and dissociation via an efficient one (at sites Y, Y') become possible.