To study the structural influence of the DNA phosphate backbone on cleavage and binding affinity of E. coli topoisomerase I, oligonucleotides were synthesized with a chiral phosphorothioate replacing the non-bridging oxygens at each position along the backbone of dA7. A deoxy-iodo-uracil replaced the 5Õ base to crosslink by UV and assess binding affinity. At the scissle phosphate there was little effect. At the +1 position, the Rp- thio-substitution reduced the cleavage rate by a factor of 10. At positions +3 and Ð2 from the scissle bond the Rp-isomer was cleaved at a faster rate than the Sp-isomer. Observed differences in binding affinity agreed with the observed rate differences, except at the Ð2 position. This demonstrates the importance of backbone contacts in the active site of E. coli topoisomerase I. Contacts within the active site of the protein were identified by uv-crosslinking and by partial digestion with trypsin. The contact sites identified were the zinc binding domain and the sites reported in the recent crystal structure of the 67kDa N-terminus of this enzyme and nucleotides¹

1. Feinberg, H. et. al. , Nature Struc. Biol. (1999) 6, 918.