Transposon-mediated site-specific recombination in vitro: DNA cleavage and protein-DNA linkage at the recombination site

Resolvase, the product of the tnpR gene of the transposable element γδ, mediates a site-specific recombination between two copies of the element directly repeated on the same replicon. The resolution site, res, at which resolvase acts lies in the intercistronic region between the tnpA and tnpR genes. We have studied this site-specific recombination in vitro. In the absence of Mg 2+, a resolvase- res complex is formed, which contains DNA molecules that have been cleaved at res. Our data suggest that in this complex resolvase is covalently attached to the 5′ ends of the cleaved DNA, leaving free 3′ hydroxyl groups. DNA cleavage is stimulated by the interaction of two res sites on the same substrate molecule and appears to be an intermediate step in normal res site recombination. We show that the DNA is cut within a region previously identified as containing the crossover point at the palindromic sequence 5′− TT AT ↓AA AA ↑TA TT to generate 3′ extensions of two bases.