NOVEL-APPROACH TO MOLECULAR-CLONING AND POLYNUCLEOTIDE SYNTHESIS USING VACCINIA DNA TOPOISOMERASE

Construction of chimaeric DNA molecules in vitro relies traditionally on two enzymatic steps catalyzed by separate protein components. Site- specific restriction endonucleases are used to generate linear DNAs wi th defined termini that can then be joined covalently at their ends vi a the action of DNA ligase. A novel approach to the synthesis of recom binant DNAs exploits the ability of a single enzyme, vaccinia DNA topo isomerase, to both cleave and rejoin DNA strands with extreme specific ity at each step. Placement of the CCCTT cleavage motif for vaccinia t opoisomerase near the end of a duplex DNA permits efficient generation of a stable, highly recombinogenic protein-DNA adduct that can religa te only to acceptor DNAs that contain complementary single-strand exte nsions. Linear DNAs containing CCCTT cleavage sites at both ends (biva lent substrates) can be activated by topoisomerase and inserted into a plasmid vector in a simple and rapid in vitro procedure that is espec ially well suited to the molecular cloning of polymerase chain reactio n-amplified DNAs. Activation of polyvalent (e.g. branched) DNA substra tes by topoisomerase offers a potentially powerful method for the synt hesis of two- and three dimensional polynucleotide networks.