Replicative resolution of integron cassette insertion

Abstract

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.

Integrons: Past, Present, and Future

“…3B) serves to orient the polarity of the recombination event by determining which strand is recombined and thus ensures that cassettes are inserted in the correct orientation (2,72). Recombination between attI and attC involves only the bottom strand of the incoming attC, and the singlestranded recombination structure is then resolved by replication (73,74). Because IntI activity is dependent on structure, rather than sequence, this explains why diverse IntI proteins are able to mobilize gene cassettes with very different attC sequences (75).…”

Bacterial Genome Instability

“…The attC recombination site is a cassette-specific imperfect inverted repeat, variable in length and sequence, that can form secondary structures through self-pairing of the DNA strands (191). The IntI integrase recognizes the secondary structure of the bottom strand of the single-stranded attC site on the gene cassette and recombines it with the attI site in the integron (191)(192)(193), inserting the gene cassette just behind the P c promoter in an orientation that usually results in the expression of the newly integrated gene. Integration of a single-stranded product may favor events following DNA transfer of a single strand via conjugation (i.e., in an intercellular event).…”

The Integron: Adaptation On Demand

“…Resolution of the junction is then carried out by a replicative process without the involvement of a second strand exchange of any kind ( Fig. 3) (54). Therefore, cassette insertion is a semiconservative process and implies the release of the initial attI-containing substrate.…”