Wild-type Flp recombinase cleaves DNA in trans.

Site-specific recombinases of the Integrase family utilize a common chemical mechanism to break DNA strands during recombination. A conserved Arg-His-Arg triad activates the scissile phosphodiester bond, and an active-site tyrosine provides the nucleophile to effect DNA cleavage. Is the tyrosine residue for the cleavage event derived from the same recombinase monomer which provides the RHR triad (DNA cleavage in cis), or are the triad and tyrosine derived from two separate monomers (cleavage in trans)? Do all members of the family follow the same cleavage rule, cis or trans? Solution studies and available structural data have provided conflicting answers. Experimental results with the Flp recombinase which strongly support trans cleavage have been derived either by pairing two catalytic mutants of Flp or by pairing wild-type Flp and a catalytic mutant. The inclusion of the mutant has raised new concerns, especially because of the apparent contradictions in their cleavage modes posed by other Int family members. Here we test the cleavage mode of Flp using an experimental design which excludes the use of the mutant protein, and show that the outcome is still only trans DNA cleavage.