MECHANISM OF INHIBITION OF VACCINIA DNA TOPOISOMERASE BY NOVOBIOCIN AND COUMERMYCIN

Vaccinia DNA topoisomerase, a eukaryotic type I enzyme, has unique pha rmacological properties, including sensitivity to the coumarin drugs n ovobiocin and coumermycin, which are classical inhibitors of DNA gyras e, a type II enzyme. Whereas coumarins inhibit gyrase by binding the G yrB subunit and thereby blocking the ATP-binding site, they inhibit va ccinia topoisomerase by binding to the protein and blocking the intera ction of enzyme with DNA. Noncovalent DNA binding and single-turnover DNA cleavage by topoisomerase are inhibited with K-I values of 10-25 m u M for coumermycin and 350 mu M for novobiocin. Spectroscopic and flu orescence measurements of drug binding to enzyme indicate a single bin ding site on vaccinia topoisomerase for coumermycin (K-D = 27 +/- 5 mu M) and two classes of binding sites for novobiocin, one tight site (K -D1 = 20 +/- 5 mu M) and several weak sites (K-D2 = 513 +/- 125 mu M; n = 4.9 +/- 0.7). Addition of a stoichiometric amount of DNA to a pref ormed coumermycin-topoisomerase complex quantitatively displaces the d rug, indicating that coumermycin binding and DNA binding to topoisomer ase are mutually exclusive. A simple interpretation is that the site o f drug binding coincides or overlaps with the DNA-binding site on the topoisomerase. Both novobiocin and coumermycin alter the susceptibilit y of vaccinia topoisomerase to proteolysis with either chymotrypsin or trypsin; similar effects occur when topoisomerase binds to duplex DNA .