Topological selectivity of a hybrid site-specific recombination system with elements from Tn3 res/resolvase and bacteriophage P1 loxP/Cre

Ln order to investigate the functions of the parts of the Tn3 recombination site res, we created hybrid recombination sites by placing the loxP site f or Cre recombinase adjacent to the "accessory" resolvase-binding sites II a nd III of res. The efficiency and product topology of in vitro recombinatio n by Cre between two of these hybrid sites were affected by the addition of Tn3 resolvase. The effects of resolvase addition were dependent on the rel ative orientation and spacing of the elements of the hybrid sites. Substrat es with sites II and III of res close to loxP gave specific catenated or kn otted products (four-noded catenane, three-noded knot) when resolvase and C re were added together. The product topological complexity increased when t he length of the spacer DNA segment between loxP and res site II was increa sed. Similar resolvase-induced effects on Cre recombination product topolog y were observed in reactions of substrates with loxP sites adjacent to full res sites. The results demonstrate that the res accessory sites are suffic ient to impose topological selectivity on recombination, and imply that int ertwining of two sets of accessory sites defines the simple catenane produc t topology in normal resolvase-mediated recombination. They are also consis tent with current models for the mechanism of catalysis by Cre. (C) 1999 Ac ademic Press.