# 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
Interestingly, in silico analyses reveal that _attC\({}_{\text{bs}}\)_ in SCI are predominantly carried on the leading strand template and that there is a correlation between the cassette array orientation and its respective size [74]. Indeed, the few SCI identified in inverse orientation, found exclusively in _Xanthomonas_ species, present a number of cassettes that do not exceed 22 cassettes, while those found in _Vibrio_ in the opposite orientation can reach up to 200 cassettes (217 in _Vibrio vulnificus_). This bias of orientation in SCI, by limiting the cassette excision rate, could avoid cassette loss and ensure a large reservoir of genetic functions that will stay accessible for their MI counterparts. On the other hand, the _attC_ sites could be a target for several host factors and more precisely for the proteins able to bind single-stranded DNA such as the _E. coli_ Single-Stranded DNA-Binding protein (SSB). SSB regulates many DNA processes such as replication, reparation, and homologous recombination [75]. SSB can spontaneously migrate along ssDNA. This diffusion helps in the local displacement of SSB by an elongating RecA filament. SSB diffusion also melts short DNA hairpins transiently and stimulates RecA filament elongation on DNA with secondary structures [76]. We precisely demonstrated that SSB is able to strongly bind folded _attC_ sites and to destabilize them. The SSB effect is observed only in absence of the integrase. Indeed, when the integrase is expressed, the SSB overexpression or inactivation do not impact the recombination reaction mediated by the integrase [77]. Thus, the integrase is able to counterbalance the observed effect of SSB on _attC_ site folding. The integrase possesses an intrinsic property to capture _attC_ sites at the moment of their extrusion, stabilizing them and recombining them efficiently (Figure 5). The precise mechanism of action of SSB was elucidated using an in vitro approach based on single-molecule Forster Resonance Energy Transfer. The results showed that _attC_ hairpins have a conserved high GC-content near their apical loop, enabling SSB to open the hairpin and create a dynamic equilibrium between _attC_ fully opened by SSB and a partially structured _attC_-6-SSB complex. This latter complex is recognized by the integrase IntI, which extrudes the full hairpin upon binding while displacing SSB [78]. During this process, the SSB-flattened _attC_ sites are converted in IntI-bound recombinogenic forms. In conclusion, we demonstrated that SSB, which hampers _attC_ site folding in the absence of the integrase, likely plays an important role in maintaining the integrity of the _attC_ sites and thus the recombinogenic functionality of the integron _attC_ sites (Figure 5). Indeed, the stability of DNA secondary structures in the chromosome must be controlled and restrained. Too stable secondary structures could be deleted by slippage or by action of the SbcCD complex, which cleavesDNA hairpins [70]. Moreover, such structures could be toxic for the cell by blocking the replication progression [79].