ESCHERICHIA-COLI TOPOISOMERASE-IV - PURIFICATION, CHARACTERIZATION, SUBUNIT STRUCTURE, AND SUBUNIT INTERACTIONS

DNA sequence analysis of Escherichia coli parC and parE, encoding the subunits of topoisomerase IV (Topo IV) (Kato, J.-I., Suzuki, H., and I keda, H. (1992) J. Biol. Chem. 267, 25676-25684), showed that ParC was 22 amino acids longer on the N terminus and ParE was 29 amino acids l onger on the C terminus than reported previously. E. coli strains bear ing bacteriophage T7 RNA polymerase-based expression plasmids carrying both intact and truncated parC and parE were used to overproduce the ParC and ParE proteins. Full-length ParC and ParE were required to rec onstitute Topo IV activity, whereas the truncated ParC and ParE were i nactive. Topo IV activity was supported only by ATP or dATP. The [ATP] 1/2 for DNA relaxation was 0.45 mM, almost 25-fold higher than the [AT P]1/2 for decatenation of kinetoplast DNA. Topo IV activity was inhibi ted by the quinolone and coumarin antibiotics, although the concentrat ions required for 50% inhibition of activity were 3-30-fold higher tha n those required to inhibit DNA gyrase. The norfloxacin-induced DNA cl eavage patterns of Topo IV and DNA gyrase were distinct but overlappin g. The native forms of ParC and ParE were a dimer and a monomer, respe ctively; whereas the active form of Topo IV was a heterotetramer, ParC 2ParE2. The inactivity of the truncated forms of ParC and ParE could b e attributed to their failure to form the heterotetramer.