Structural heterogeneity of attC integron recombination sites revealed by optical tweezers

摘要

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 single-stranded 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 single-molecule optical tweezers (OT) to characterize secondary structures formed by the attC bottom (?|${{att}}{{{C}}_{{m{bs}}}}$|?) and top (?|${{att}}{{{C}}_{{m{ts}}}}$|?) 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 |${{att}}{{{C}}_{{m{bs}}}}$| predominantly formed the straight hairpin, while the |${{att}}{{{C}}_{{m{ts}}}}$| 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 |${{att}}{{{C}}_{{m{ts}}}}$|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.