Effect of phosphorothioate substitutions on DNA cleavage by Escherichia coli DNA topoisomerase I

Abstract

To evaluate the structural influence of the DNA phosphate backbone on the activity of Escherichia coli DNA topoisomerase I, modified forms of oligonucleotide dA 7 were synthesized with a chiral phosphorothioate replacing the non-bridging oxygens at each position along the backbone. A deoxy-iodo-uracil replaced the 5′-base to crosslink the oligonucleotides by ultraviolet (UV) and assess binding affinity. At the scissile phosphate there was little effect on the cleavage rate. At the +1 phosphate, the rectus phosphorus ( Rp)-thio-substitution reduced the rate of cleavage by a factor of 10. At the +3 and −2 positions from the scissile bond, the Rp-isomer was cleaved at a faster rate than the sinister phosphorus ( Sp)-isomer. The results demonstrate the importance of backbone contacts between DNA substrate and E. coli topoisomerase I.