Structural heterogeneity of attC integron recombination sites revealed by optical tweezers

Abstract

A predominant tool for adaptation in Gram-negative bacteria is the functional genetic platform called integron. Integrons capture and rearrange promoterless gene cassettes in a unique recombination process involving the recognition of folded singlestranded DNA hairpins-so-called attC sites-with a strong preference for the attC bottom strand. While structural elements have been identified to promote this preference, their mechanistic action remains incomplete. Here, we used high-resolution singlemolecule optical tweezers (OT) to characterize secondary structures formed by the attC bottom (attCbs) and top (attCts) strands of the paradigmatic attCaadA7 site. We found for both sequences two structures-a straight, canonical hairpin and a kinked hairpin. Remarkably, the recombination-preferred attCbs predominantly formed the straight hairpin, while the attCts preferentially adopted the kinked structure, which exposes only one complete recombinase binding box. By a mutational analysis, we identified three bases in the unpaired central spacer, which could invert the preferred conformations and increase the recombination frequency of the attCts in vivo. A bioinformatics screen revealed structural bias toward a straight, canonical hairpin conformation in the bottom strand of many antibiotic resistance cassettes attC sites. Thus, we anticipate that structural fine tuning could be a mechanism in many biologically active DNA hairpins.