Alteration of DNA Primary Structure by DNA Topoisomerase I. Isolation of the Covalent Topoisomerase I−DNA Binary Complex in Enzymatically Competent Form

Abstract

DNA ligation by DNA topoisomerase I was investigated employing synthetic DNA substrates containing a single strand nick. Site-specific cleavage of the DNA by topoisomerase I in proximity to the nick resulted in uncoupling of the cleavage and ligation reactions of the enzyme, thereby trapping the covalent enzyme−DNA intermediate. DNA cleavage could be reversed by the addition of acceptor oligonucleotides containing a free 5‘-OH group and capable of hybridizing to the noncleaved strand of the “suicide substrates”. Utilizing acceptors with partial complementarity, modification of nucleic acid structure has been obtained. Modifications included the formation of DNA insertions, deletions, and mismatches. To further evaluate the potential of topoisomerase I to mediate structural transformations of DNA, acceptor oligonucleotides containing nucleophiles other than OH groups at the 5‘-end were studied as substrates for the topoisomerase I-mediated ligation reaction. Toward this end, oligonucleotides containing 5‘-...