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

To evaluate the structural influence of the DNA phosphate backbone on the a ctivity of Escherichia coli DNA topoisomerase L modified forms of oligonucl eotide dA(7) were synthesized with a chiral phosphorothioate replacing the non-bridging oxygens at each position along the backbone. A deoxy-iodo-urac il replaced the 5 ' -base to crosslink the oligonucleotides by ultraviolet (UV) and assess binding affinity. At the scissile phosphate there was littl e effect on the cleavage rate. At the + I phosphate, the rectus phosphorus (Rp)-thio-substitution reduced the rate of cleavage by a factor of 10. At t he + 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 demo nstrate the importance of back-bone contacts between DNA substrate and E. c oli topoisomerase I. (C) 2001 Published by Elsevier Science BN.