Topo (cloning technology)

The key to TOPO® Cloning is the enzyme DNA topoisomerase I, which functions as both a restriction enzyme and a ligase. Its biological role is to cleave and rejoin DNA during replication. To harness the religating activity of topoisomerase, vectors are provided linearized with topoisomerase I covalently bound to each 3' phosphate.

Vaccinia virus topoisomerase I specifically recognizes the pentameric sequence 5´-(C/T)CCTT-3´ and forms a covalent bond with the phosphate group of the 3´ thymidine. It cleaves one DNA strand, enabling the DNA to unwind. The enzyme then religates the ends of the cleaved strand and releases itself from the DNA.