TY - JOUR
T1 - Mechanism of inhibition of vaccinia DNA topoisomerase by novobiocin and coumermycin
AU - Sekiguchi, Joann
AU - Stivers, James T.
AU - Mildvan, Albert S.
AU - Shuman, Stewart
PY - 1996/1/26
Y1 - 1996/1/26
N2 - Vaccinia DNA topoisomerase, a eukaryotic type I enzyme, has unique pharmacological properties, including sensitivity to the coumarin drugs novobiocin and coumermycin, which are classical inhibitors of DNA gyrase, a type II enzyme. Whereas coumarins inhibit gyrase by binding the GyrB subunit and thereby blocking the ATP-binding site, they inhibit vaccinia topoisomerase by binding to the protein and blocking the interaction of enzyme with DNA. Noncovalent DNA binding and single-turnover DNA cleavage by topoisomerase are inhibited with K(I) values of 10-25 μM for coumermycin and 350 μM for novobiocin. Spectroscopic and fluorescence measurements of drug binding to enzyme indicate a single binding site on vaccinia topoisomerase for coumermycin (K(D) = 27 ± 5 μM) and two classes of binding sites for novobiocin, one tight site (K(D1) = 20 ± 5 μM) and several weak sites (K(D2) = 513 ± 125 μM; n = 4.9 ± 0.7). Addition of a stoichiometric amount of DNA to a preformed coumermycin-topoisomerase complex quantitatively displaces the drug, indicating that coumermycin binding and DNA binding to topoisomerase are mutually exclusive. A simple interpretation is that the site of drug binding coincides or overlaps with the DNA-binding site on the topoisomerase. Both novobiocin and coumermycin alter the susceptibility of vaccinia topoisomerase to proteolysis with either chymotrypsin or trypsin; similar effects occur when topoisomerase binds to duplex DNA.
AB - Vaccinia DNA topoisomerase, a eukaryotic type I enzyme, has unique pharmacological properties, including sensitivity to the coumarin drugs novobiocin and coumermycin, which are classical inhibitors of DNA gyrase, a type II enzyme. Whereas coumarins inhibit gyrase by binding the GyrB subunit and thereby blocking the ATP-binding site, they inhibit vaccinia topoisomerase by binding to the protein and blocking the interaction of enzyme with DNA. Noncovalent DNA binding and single-turnover DNA cleavage by topoisomerase are inhibited with K(I) values of 10-25 μM for coumermycin and 350 μM for novobiocin. Spectroscopic and fluorescence measurements of drug binding to enzyme indicate a single binding site on vaccinia topoisomerase for coumermycin (K(D) = 27 ± 5 μM) and two classes of binding sites for novobiocin, one tight site (K(D1) = 20 ± 5 μM) and several weak sites (K(D2) = 513 ± 125 μM; n = 4.9 ± 0.7). Addition of a stoichiometric amount of DNA to a preformed coumermycin-topoisomerase complex quantitatively displaces the drug, indicating that coumermycin binding and DNA binding to topoisomerase are mutually exclusive. A simple interpretation is that the site of drug binding coincides or overlaps with the DNA-binding site on the topoisomerase. Both novobiocin and coumermycin alter the susceptibility of vaccinia topoisomerase to proteolysis with either chymotrypsin or trypsin; similar effects occur when topoisomerase binds to duplex DNA.
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U2 - 10.1074/jbc.271.4.2313
DO - 10.1074/jbc.271.4.2313
M3 - Article
C2 - 8567695
AN - SCOPUS:0030070923
SN - 0021-9258
VL - 271
SP - 2313
EP - 2322
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 4
ER -