Site-specific Recombination at resSites Containing DNA-binding Sequences for both Tn21 and Tn3 Resolvases

Abstract

Tn3 and *gamma;δ resolvases catalyse site-specific recombination at ressites from Tn3 but not at Tn21 ressites. Tn21 resolvase has no activity at Tn3 and acts only at Tn21 sites. In both Tn3 and Tn21, reshas three binding sites for the cognate resolvases; the cross-over site, I; and the accessory sites II and III, from which the bound proteins may stabilize the synaptic complex by protein-protein interactions. In this study hybrid ressites were made by replacingb either II or III in the Tn21 ressite with the equivalent sequence from Tn3. Plasmids containing either a hybrid and a wild-type Tn21 ressite, or two hybrid sites, were tested for recombination. Relative to the reaction with two wild-type sites, recombination by Tn21 resolvase was reduced by replacing II at one ressite and it was reduced further by replacing II at both loci but, in both cases, Tn21 recombination was enhanced by Tn3 or γδ resolvases. Very few of the amino acid on the external surface of γδ resolvases are conserved in Tn21. Moreover, mutants of γδ resolvase with defective protein-protein interactions also enhanced Tn21 recombination at this hybrid site. The resolvase at II thus seems not to be involved in protein-protein interactions and its main role may be to bend the DNA to the required structure. The replacement of III in the Tn21 site with Tn3 sequence also reduced recombination by Tn21 resolvase, especially when both loci carried the alteration but, in contrast to before, Tn3 or γδ resolvases now inhibited the Tn21 reaction. Recombination thus seems to require identical proteins at I and III, perhaps to allow for protein-protein interactions.