# Unbridled Integons: A Matter of Host Factors
## 5 Host Factors Influencing the Recombination of the Single-Stranded _attC_ Substrates
### Resolution of the Atypical Holliday Junction  
#### 5.2.1 The Replication Process Is Involved in the aHJ Resolution  
Site-specific recombination catalyzed by tyrosine recombinases follows an archetypical pathway consisting of two consecutive strand exchanges. The first strand exchange generates a Holliday junction, which is resolved by a second strand exchange. As said above, intergon integrases are atypical Y-recombinases since they recognize and recombine double-stranded _attI_ site with a folded single-stranded _attC_ site, the bottom one. A consequence of this _ss attC_ specificity is that the _attI_\(\times\)_attC_ reaction, which leads to cassette insertion, involves both dsDNA (_attI_) and ssDNA (_attC_), thus resulting, after the first strand exchange, in the formation of an atypical Holliday junction (aHJ) (Figure 6). Due to the asymmetry of this complex, a second strand exchange on the _attC_ bottom strand would form linearized abortive recombination products. We demonstrated that aHJ resolution would rely on a mechanism involving the host machinery of replication [87]. 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_ recombinogenic bottom strand, is exchanged. Indeed, the recombination products contain the _attC_ site bottom strand and its entire de novo synthesized complementary strand. Therefore, we demonstrated the replicative resolution of single strand recombination in integrons and ruled out the involvement of a second strand exchange of any kind in the _attC_\(\times\)_attI_ reaction.  
Figure 6: Replicative resolution pathway during cassette insertion in _attI_ site. Both substrates (_attI_-containing replicon and the cassette) are presented by purple lines, and the single-stranded _attC_ bottom strand (ss _attC_\({}_{\text{Rs}}\)) and double-stranded _attI_ (ds _attI_) sites are represented, respectively, by green and red lines. Note that the top strand of the _attC_ site is represented as a dotted line because we do not know the nature of the cassettes (ss or ds). Synaptic complexes during the first strand cleavage and transfer are shown. Only one strand from each duplex is cleaved and transferred forming an atypical Holliday junction (aHJ) due to the single-stranded nature of the _attC_ site. Each time, the four integrase protomers are shown. The two activated protomers are represented by dark gray ovals and the inactive ones by light gray ovals. The aHJ resolution implies a replication step. The origin of replication is represented by a purple circle and the newly synthesized leading and lagging strands by dashed purple lines. Both products are represented: the inserted cassette results from the bottom strand replication and the initial _attI_-containing replicon from the top strand replication. Hybrid _attC_ and _attI_ sites are indicated.  
Note that the nature of host factors is not yet completely known because we do not know if the resolution occurs by the passage of the replication fork and/or by the intervention of host factors restarting stalled replication forks such as the PriA helicase [88]. We also studied the recombination between two _attl_ sites, a reaction involving exclusively double-stranded substrates, for which the second exchange of strands is not abortive. Using genetic approaches and high-throughput sequencing, we demonstrated in vivo that both strands of the _attl_ site are reactive and prove that the resolution of this HJ can follow either the replicative pathway distinctive of the integrin integrase (see above) or the second strand exchange characteristic of Y-recombinases [89].  
Altogether, these results demonstrated that integrases have evolved towards new recombination substrates (i.e., single-stranded) and resolution pathways (i.e., replicative way) connecting the integrin with the cell physiology.