# Unbridled Integons: A Matter of Host Factors
## 5 Host Factors Influencing the Recombination of the Single-Stranded _attC_ Substrates
### attC Site Folding Regulation
#### 5.1.2 Host Factors Involved in \(attC\) Site Folding during the Double-Stranded Pathway  
\(attC\) sites can be folded from dsDNA. Indeed, inverted repeats (perfect or imperfect) have the potential to form branched structures called cruciforms, in which inter-strand base pairing within the symmetric region is replaced by intra-strand base pairing [80]. The formation of a cruciform involves a great deal of structural disruption as it requires a complete reorganization in base pairing. We demonstrated that ability of \(attC\) sites to be extruded depends on two parameters: first, the length of their VTS since to go from a ds state to a cruciform, an \(attC\) site needs to melt at least the length of its VTS; and second, their propensity to form non-recombingenic structures [72]. In summary, only \(attC\) sites with small VTS (e.g., \(attC_{\mathit{anad}A\mathit{Z}}\), \(attC_{\mathit{CerrA2}}\)... ) and low propensity to form non-recombingenic structure could be efficiently extruded. In addition to these two parameters, we demonstrated that DNA superhelicity influences the \(attC\) site extrusion in vitro and in vivo [72]. Indeed, cruciforms have lower thermodynamic stability than the \"classic\" double-stranded DNA. They have been observed only in negatively supercoiled molecules, in which the unfavorable free energy of formation is offset by the topology of the torsionally stressed molecule. In agreement with these observations, we demonstrated that proteins increasing negative superhelicity such as gyrase and topoisomerase directly favor cruciform extrusion of \(attC\) sites and their recombination [72]. Precisely, we demonstrated that in a \(topA10\)\(gyrB266\) strain, \(attC\) sites recombine about 10 times less efficiently than in a WT strain. By extension, these results mean that factors expected to influence the superhelicity in bacteria could modify the rate of cassette recombination. Replication, transcription, mutations in certain genes (such as those encoding RNA polymerase), HU, HNS, salt shock, high osmolarity, nutrient downshift, infections by phages and late phase of growth: all these factors influence the local superhelicity [81; 82; 83; 84]. The level of superhelicity can also vary between bacterial strains. Indeed, the average supercoil density of a pBR reporter plasmid extracted from mid-log cultures of _Salmonella_ is 13% lower than that from _E. coli_[85]. Interestingly, topology analysis of reporter plasmids also revealed higher levels of negative supercoiling in strains with the constitutively expressed SOS network, suggesting a link between the induction of SOS response and changes in DNA topology [86]. The network  
Figure 5: Model of the SSB protein flattening the folded \(attC\) sites. The model shows how SSB opens the \(attC\) hairpin via the \(attC\)-6–SSB state, followed by the SSB-65 and SSB-35 modes, ensuring the integrity of the \(attC\) sites in absence of integrase (Intl). The integrase acts by capturing the \(attC\) sites at the moment of their extrusion, preventing the melting effect of SSB. Thus, SSB is released and the \(attC\) folded structure is stabilized.  
of _attC_ site regulation linked to the superhelicity is therefore considerable and has not yet been completely explored.