DNA duplexes containing 3′-deoxynucleotides as substrates for DNA topoisomerase I cleavage and ligation

The DNA cleavage-ligation reaction of DNA topoisomerase I was investigated employing synthetic DNA substrates containing 3'-deoxyadenosine or 3'-deoxythymidine at specific sites and acceptor oligonucleotides of different lengths. The modified nucleotides were substituted systematically within the putative enzyme-binding domain and also next to the high efficiency cleavage site to determine the effect of single base changes on enzyme function. Depending on the site of substitution, the facility of the cleavage and ligation reactions were altered. The bases at positions -1 and -2 on the noncleaved strand were found to be important for determining the site of cleavage. Inclusion of 3'-deoxythymidine in the scissile strand at position -1 permitted the demonstration that topoisomerase I can cleave and form a 2' --> 5'-phosphodiester linkage. Partial duplexes doubly modified at positions -4 or -6 in the noncleaved strand and at positions +1 or -1 within scissile strand were not good substrates for topoisomerase I, showing that cleavage can depend importantly on binding interactions based on structural alterations at spatially separated sites. Substitution of a 3'-deoxynucleotide on the scissile strand at position -6 enhanced formation of the ligation product resulting from cleavage at site 1 and suppressed cleavage at site 2.