# Unbridled Integons: A Matter of Host Factors
## 5 Host Factors Influencing the Recombination of the Single-Stranded _attC_ Substrates
### attC Site Folding Regulation
#### 5.1.1 Host Factors Involved in the _attC_ Site Folding during the Single-Stranded Pathway  
In several bacterial cellular processes, DNA can be single-stranded, for instance, during replication; during DNA repair; or, more importantly, during rolling-circle replication, bacterial conjugation, natural transformation, and infection by some viruses. We previously investigated some of them, such as conjugation, natural transformation, and replication, and demonstrated that they favor the proper folding of _attC_ sites. During conjugation, ssDNA is unwound from the duplex plasmid, actively transferred into a recipient neighboring bacterium, and recircularized (for review, see [71]). We demonstrated that, by this way, conjugation ensures the folding of _attC_ sites and that most of the _attC_ sites displayed similar recombination frequencies when delivered by conjugation, regardless of their size or VTS length. Indeed, VCR sites with large VTS and _attC_ sites with shorter VTS (e.g., _attC\({}_{\text{nadA7}}\)_) are recombined with similar efficiency (Around \(10^{-3}\), [72]). On the contrary, in replicative recombination conditions, a significant correlation was observed between the recombination frequencies and the VTS length of _attC_ sites (i.e., up to 4-log of decrease using _attC_ sites with long VTS, [72]). The cassette recruitment is also facilitated by the ssDNA delivery during the transformation process [56]. Up to now, it was demonstrated that natural transformation mediates uptake and exchange of free ssDNA when sufficient  
Figure 4: _attC_ site folding pathways. The different possible cellular pathways allowing proper folding of the _attC_ sites are shown. The _attC_ sites can be folded during the single-stranded DNA pathway (1) when delivered during replication, conjugation, phage infection, and transformation. They can also be folded from supercoiled double-stranded DNA (2) as cruciform structures. IntI and SSB monomers are, respectively, represented as grey ovals and grey circles. The origin of replication is represented by a purple circle and the newly synthesized leading and lagging strands by dashed purple lines. homology is present between the incoming DNA and the bacterial genome [73]. We demonstrated that integration of genetic material during natural transformation can be also mediated by homology independent DNA mechanisms, notably by recombining gene cassettes inside integrins in an integrase dependent way. Hence, HGT not only contributes to the transfer of cassettes, it is also a perfect route for recruitment by integrins simply because it favors the folding of recombingenic _attC_ sites. This allows one to couple cassette recombination in bacteria with the moment of their entry under a single-stranded DNA form. Another source of single-stranded DNA in the cell is the replication process. We also demonstrated that replication favors _attC_ site folding [72]. During replication, one of the two new strands, the lagging strand, is made as discontinuous DNA pieces, and the corresponding template strand contains large single-stranded regions (i.e., between Okazaki fragments). We demonstrated that when the bottom recombinogenic strand of the _attC_ site (_attC\({}_{\text{bs}}\)_) is located on the lagging strand template, its folding is favored. This has been proven by cloning an _attC_ site in both orientations toward replication in a unidirectionally replicated plasmid and comparing their respective integration rate. The results showed that higher availability of ssDNA in the lagging strand template impacts the frequency of _attC_\(\times\)_att1_ recombination [72]. We also showed that the orientation of the cassette array relative to replication regulates the cassette excision rate (i.e., _attC_\(\times\)_attC_ recombination), by influencing the _attC_ site folding [74].