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We recently showed that cassette integration and deletion in integron platforms were occurring through unconventional site-specific recombination reactions involving only the bottom strand of  attC  sites. The lack of sequence conservation among  attC  sites led us to hypothesize that sequence-independent structural recognition determinants must exist within  attC  sites. The structural data obtained from a synaptic complex of the  Vibrio cholerae  integrase with the bottom strand of an  attC  site has shown the importance of extra helical bases (EHB) inside the stem-loop structure formed from the bottom strand. Here, we systematically determined the contribution of three structural elements common to all known single-stranded  attC  site recombination substrates (the EHBs, the unpaired central spacer (UCS), and the variable terminal structure (VTS)) to strand choice and recombination. Their roles have been evaluated  in vivo  in the  attI × attC  reaction context using the suicide conjugation assay we previously developed, but also in an  attC × attC  reaction using a deletion assay. Conjugation was used to deliver the  attC  sites in single-stranded form. Our results show that strand choice is primarily directed by the first EHB, but the presence of the two other EHBs also serves to increase this strand selection. We found that the structure of the central spacer is essential to achieve high level recombination of the bottom strand, suggesting a dual role for this structure in active site exclusion and for hindering the reverse reaction after the first strand exchange. Moreover, we have shown that the VTS has apparently no role in strand selectivity.

Structural Features of Single-Stranded Integron Cassette attC Sites and Their Role in Strand Selection