SITE-SPECIFIC RECOMBINATION AT RES SITES CONTAINING DNA-BINDING SEQUENCES FOR BOTH TN21 RESOLVASE AND CAP

The res sites, the loci for site-specific recombination by resolvase, contain three binding sites for the protein; the cross-over site, I, a nd accessory sites II and In. The role of DNA bending by resolvase was examined by replacing either II or III in the res site from Tn21 with the recognition sequence for a heterologous DNA-bending; protein, CAP (the catabolite-gene activator protein from Escherichia coli). The CA P sequence was placed at either the same position as the target sequen ce for Tn21 resolvase or a different position along the DNA. The activ ity of Tn21 resolvase for recombination between each hybrid and a wild -type res site was measured in the presence of CAP and cyclic AMP. Whe n III was substituted, CAP inhibited Tn21 recombination, except when t he CAP sequence was placed sufficiently far away from site II to allow resolvase to bind non-specifically to the DNA between II and the CAP site. With the substitutions at II, the extent of Tn21 recombination i n the presence of CAP varied with the position of the CAP sequence: mo re recombination was observed when it superimposed the target sequence for resolvase than when it was displaced by five base-pairs. Efficien t recombination by Tn21 resolvase thus seems to demand the cognate pro tein at site III in res, presumably for protein-protein interactions i n the synaptic complex, while the function of resolvase at site II can be fulfilled, at least in part, by a heterologous DNA bend.