A HOT-SPOT IN PLASMID-F FOR SITE-SPECIFIC RECOMBINATION MEDIATED BY TN21 INTEGRON INTEGRASE

Integron In2 integrase (IntI1)-mediated site-specific recombination be tween two primary sites occurs at a high frequency, while that between a primary and a secondary site occurs at frequencies around 10,000 ti mes lower. Secondary sites consist of a pentanucleotide with only two fully conserved residues (GWTMW). The analysis of IntI1-mediated recom binants in the plasmid pOX38 revealed the existence in this plasmid of a site used at a frequency intermediate between those of primary and secondary sites. Analysis of this site showed two potentially relevant structural features: first, a set of two consensus pentanucleotides, separated by 5 hp and in opposite orientations, forming what will be c alled a double site; and second, a longer sequence with some extent of sequence symmetry with the double site at its 3' end. A recombinant p lasmid, pSU18P, containing a double site was constructed. Examination of R388-pSU18P recombinants showed that double sites were used prefere ntially over single pentanucleotides by IntI1, Comparisons of the nucl eotide sequences of known 59-bp elements showed that in most cases the re was a double site at each element end. Mutagenesis of the F hot spo t was carried out to make it look more like the consensus 59-bp elemen t. The improved sites showed recombination frequencies and specificiti es almost comparable to those observed at IntI1 primary sites.