The protein-protein interactions required for assembly of the Tn3resolution synapse

The site-specific recombinase Tn3resolvase initiates DNA strand exchange when tworesrecombination sites and six resolvase dimers interact to form a synapse. The detailed architecture of this intricate recombination machine remains unclear. We have clarified which of the potential dimer-dimer interactions are required for synapsis and recombination, using a novel complementation strategy that exploits a previously uncharacterized resolvase fromBartonella bacilliformis(Bart). Tn3and Bart resolvases recognize different DNA motifs, via diverged C-terminal domains (CTDs). They also differ substantially at N-terminal domain (NTD) surfaces involved in dimerization and synapse assembly. We designed NTD-CTD hybrid proteins, and hybridressites containing both Tn3and Bart dimer binding sites. Using these components in in vivo assays, we demonstrate that productive synapsis requires a specific R interface involving resolvase NTDs at all three dimer-binding sites inres. Synapses containing mixtures of wild-type Tn3and Bart resolvase NTD dimers are recombination-defective, but activity can be restored by replacing patches of Tn3resolvaseRinterface residues with Bart residues, or vice versa. We conclude that the Tn3/Bart family synapse is assembled exclusively byRinteractions between resolvase dimers, except for the one special dimer-dimer interaction required for catalysis.