The most striking structural feature of the yeast Saccharomyces cerevisiae 2-micron DNA plasmid is the presence of two regions of 599 bp, which are precise inverted repeats (IRs) of each other. Site-specific recombination within 2-micron is catalyzed by the Flp recombinase acting at specific DNA sequences, named Flp Recognition Target sites (FRT), which lie near the centre of each IR. However, it is not known if, within the IR, other sequences than the FRT are relevant to the functionality of the Flp reaction in vivo. In the present work we highlight a different efficiency as DNA substrate, for the minimal FRT and the entire IR sequence with respect to their molecular location. The efficiency of Flp site-specific recombination between two IRs cloned in the same orientation is about one hundred times higher than that obtained when only the two FRTs are present. Differently from the FRT, the IR sequence can also interfere with gene expression, when cloned between the kanMX4 ORF and its promoter. Moreover, we demonstrated that the amount of RAD52-dependent-repair events induced by the IR as Flp-double-strand break (DSB) substrate is higher than that induced by the minimal FRT. Finally, our results reveal that the Flp-cleavage step in vivo is not dependent on synapsis of two Flp-target sites, suggesting that it may occur prior to the DNA pairing step in the molecular process of recombination.
