(Fig. 1a).The attI site, like other site-specific recombinase binding sites, contains a core of ell as two sive towardsThe attC sites in contrast display poor sequence con gions of sequence similarity at their boundaries. These conserved regions are separated by a stretch of imperfect internal dyad symmetry 7 .One might have expected that excision of a gene cassette would occur via the classic model of Holliday junction (HJ) formation and resolution using two duplex attC sites as observed with Cre-mediated loxP recombination 10 (Fig. 1b) Structure of VchIntIA RecombinaseVchIntIA folds into two distinct domains ( le stranded attC substrates, we demonstrated that the bottom strand of attC recombined with a resident attI at a rate of 1000-fold higher than the comparable top strand of attC 12 .Disruption of the postulated 13-16 secondary structure of attC affected recombination.Based on these results we proposed a recombination model (Fig. 1c). The structure of the across the synaptic interface. This interaction allows helix I 2 from the atta unit(s) to form several important DNA contacts in trans holding the sThese trans-interactions mediated by G20'' result in only a two-fold symm synapse which may inhibit HJ isomerization as discussed below. Subunits 2 (Fig. 2b, 3). The spacing and geometry of the bases in the central region duplex preclude intersubunit interaction between the N-termini on the sam 2a, 3). The more extensive intersubunit interface across the synapse burie accessible surface area. These interfacial contacts are due namely the Integron Site-Specific RecombinationRecently, exploiting conjugation as a medium to exclusively deliver sing 9Hal-Pasteur author manuscript pasteur-00140781, version 1VchIntIA-VCR bs complex presented here provides a structural basis for IntI mediated site-specific recombination using the bottom strand of attC as a substrate. (Fig. SF6a).This suggests a higher occupancy of only one half site and change in the effective utated ty (Fig. SF6c).I and W219I), also ision frequency. These results suggest that G20'' plays a central role in maintaining an active VchIntIA-VCR bs synapse, but that other factors namely C-terminal helix exchange and B/C and A/D interfaces also contribute to the assembly of the tetrameric synapse.A folded single-strand attC substrate during integron recombination necessitates that second strand cleavage and transfer is down-regulated relative to most members ofThe biological relevance of the observed structural aspects of our m cis-and trans-interactions observed in the VchIntIA-VCR bs complex, w using a combination of EMSA and in vivo excision assays 13 . Four di observed with wild type VchIntIa-VCR (SF6a), potentially correspond however, only a 5-fold drop in excision frequency (Fig. S 6c). Mutagene (Fig. 1c).In addition the rotation (~15 o ) of the C-terminal domains within the non-attacking subunits could also reduce the rate of second strand cleavage (Fig. SF8). This movement and flanking (R' and R'') repeats. The nucleotides, T12'' (red) and G20'...

No abstract

Integrons play a major role in the dissemination of antibiotic resistance genes among Gram-negative pathogens. Integron gene cassettes form circular intermediates carrying a recombination site, attC, and insert into an integron platform at a second site, attI, in a reaction catalyzed by an integron-specific integrase IntI. The IntI1 integron integrase preferentially binds to the 'bottom strand' of single-stranded attC. We have addressed the insertion mechanism in vivo using a recombination assay exploiting plasmid conjugation to exclusively deliver either the top or bottom strand of different integrase recombination substrates. Recombination of a single-stranded attC site with an attI site was 1000-fold higher for one strand than for the other. Conversely, following conjugative transfer of either attI strand, recombination with attC is highly unfavorable. These results and those obtained using mutations within a putative attC stem-and-loop strongly support a novel integron cassette insertion model in which the single bottom attC strand adopts a folded structure generating a double strand recombination site. Thus, recombination would insert a single strand cassette, which must be subsequently processed.