Comparison of the structure–activity relationships of the integron-associated recombination sites attI3 and attI1 reveals common features

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Abstract
Incorporation of gene cassettes into integrons occurs by IntI-mediated site-specific recombination between a 59-base element (59-be) site in the cassette and an attI site in the integron. While the 59-be sites share common features and are recognized by several different IntI recombinases, the sequences of attI sites are not obviously related and are preferentially recognized by the cognate IntI. To determine the features of attI sites that are required for recombination proficiency, the structure–activity relationships of a second attI site, the attI3 site from the class 3 integron, were examined. The attI3 site was confined to within a region consisting of 68 bp from the integron backbone and 15 bp from the adjacent cassette. This region includes four IntI3-binding sites, as assessed by gel shift and methylation interference studies. Two of the binding sites are inversely oriented and constitute a simple site that includes the recombination crossover point. The two additional binding sites appear to be directly oriented and one of them is essential for efficient recombination of the attI3 site with a 59-be, but not for recombination with a second full-length attI3 site, which occurs at 100-fold lower frequency. The fourth site enhances attI3 with 59-be recombination 10-fold. The finding that the organization and overall properties of attI3 are very similar to those of attI1 indicates that these features are likely to be common to all attI sites.