SUMMARY: Structured forms of DNA with intrastrand pairing are generated in several cellular processes and are involved in biological functions. These structures may arise on single-stranded DNA (ssDNA) produced during replication, bacterial conjugation, natural transformation, or viral infections. Furthermore, negatively supercoiled DNA can extrude inverted repeats as hairpins in structures called cruciforms. Whether they are on ssDNA or as cruciforms, hairpins can modify the access of proteins to DNA, and in some cases, they can be directly recognized by proteins. Folded DNAs have been found to play an important role in replication, transcription regulation, and recognition of the origins of transfer in conjugative elements. More recently, they were shown to be used as recombination sites. Many of these functions are found on mobile genetic elements likely to be single stranded, including viruses, plasmids, transposons, and integrons, thus giving some clues as to the manner in which they might have evolved. We review here, with special focus on prokaryotes, the functions in which DNA secondary structures play a role and the cellular processes giving rise to them. Finally, we attempt to shed light on the selective pressures leading to the acquisition of functions for DNA secondary structures.

The integron is a powerful system which, by capturing, stockpiling, and rearranging new functions carried by gene encoding cassettes, confers upon bacteria a rapid adaptation capability in changing environments. Chromosomally located integrons (CI) have been identified in a large number of environmental Gram-negative bacteria. Integron evolutionary history suggests that these sedentary CIs acquired mobility among bacterial species through their association with transposable elements and conjugative plasmids. As a result of massive antibiotic use, these so-called mobile integrons are now widespread in clinically relevant bacteria and are considered to be the principal agent in the emergence and rise of antibiotic multiresistance in Gram-negative bacteria. Cassette rearrangements are catalyzed by the integron integrase, a site-specific tyrosine recombinase. Central to these reactions is the single-stranded DNA nature of one of the recombination partners, the attC site. This makes the integron a unique recombination system. This review describes the current knowledge on this atypical recombination mechanism, its implications in the reactions involving the different types of sites, attC and attI , and focuses on the tight regulation exerted by the host on integron activity through the control of attC site folding. Furthermore, cassette and integrase expression are also highly controlled by host regulatory networks and the bacterial stress (SOS) response. These intimate connections to the host make the integron a genetically stable and efficient system, granting the bacteria a low cost, highly adaptive evolution potential “on demand”.

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. Integrons play a preponderant role in the development of multiple antibiotic resistances among Gram-negative bacteria. Their success is rooted in their unique aptitude to assemble genes through a site-specific recombination process. They have recently been shown to use an unconventional recombination pathway, which involves recombination between a canonical double stranded attI site and a uniquely folded single stranded attC substrate. Due to its complex and variable structure, which includes several unpaired regions and extrahelical nucleotides, recognition of the attC site was elusive. Here, we determined the contribution of each of the different structural elements common to many folded single stranded attC substrates, in the recognition and recombination efficiency by the integron recombinase. We found that a single specific extrahelical nucleotide is responsible for the choice between the two anti-parallel complementary strands of each attC site, an essential discrimination for guaranteeing the expression of the acquired gene by the integron platform. These studies open the way to the design of synthetic sites and the use of this powerful loose-specificity recombination system for various gene transfer applications.

The integron is a bacterial recombination system that allows acquisition, stockpiling and expression of cassettes carrying protein-coding sequences, and is responsible for the emergence and rise of multiresistance in Gram-negative bacteria. The functionality of this system depends on the insertion of promoterless cassettes in correct orientation, allowing their expression from the promoter located upstream of the cassette array. Correct orientation is ensured by strand selectivity of integron integrases for the bottom strand of cassette recombination sites (attC), recombined in form of folded single-stranded hairpins. Here, we investigated the basis of such strand selectivity by comparing recombination of wild-type and mutated attC sites with different lengths, sequences and structures. We show that all three unpaired structural features that distinguish the bottom and top strands contribute to strand selectivity. The localization of Extra-Helical Bases (EHBs) directly favors integrase binding to the bottom strand. The Unpaired Central Spacer (UCS) and the Variable Terminal Structure (VTS) influence strand selectivity indirectly, probably through the stabilization of the bottom strand and the resulting synapse due to the nucleotide skew between the two strands. These results underscore the importance of the single-stranded nature of the attC site that allows such tight control over integron cassette orientation.

Site-specific recombination catalyzed by tyrosine recombinases follows a common pathway consisting of two consecutive strand exchanges. The first strand exchange generates a Holliday junction (HJ), which is resolved by a second strand exchange. In integrons, attC sites recombine as folded single-stranded substrates. Only one of the two attC site strands, the bottom one, is efficiently bound and cleaved by the integrase during the insertion of gene cassettes at the double-stranded attI site. Due to the asymmetry of this complex, a second strand exchange on the attC bottom strand (bs) would form linearized abortive recombination products. We had proposed that HJ resolution would rely on an uncharacterized mechanism, probably replication. Using an attC site carried on a plasmid with each strand specifically tagged, we followed the destiny of each strand after recombination. We demonstrated that only one strand, the one carrying the attC bs, is exchanged. Furthermore, we show that the recombination products contain the attC site bs and its entire de novo synthesized complementary strand. Therefore, we demonstrate the replicative resolution of single-strand recombination in integrons and rule out the involvement of a second strand exchange of any kind in the attC × attI reaction.