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 Ikeda, 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 longer on the C terminus than reported previously. E. coli strains bearing 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 reconstitute Topo IV activity, whereas the truncated ParC and ParE were inactive. 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 [ATP]1/2 for decatenation of kinetoplast DNA. Topo IV activity was inhibited by the quinolone and coumarin antibiotics, although the concentrations required for 50% inhibition of activity were 3-30-fold higher than those required to inhibit DNA gyrase. The norfloxacin-induced DNA cleavage patterns of Topo IV and DNA gyrase were distinct but overlapping. The native forms of ParC and ParE were a dimer and a monomer, respectively; whereas the active form of Topo IV was a heterotetramer, ParC2ParE2. The inactivity of the truncated forms of ParC and ParE could be attributed to their failure to form the heterotetramer.
Peng
H
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Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, New York.
Marians
K J
KJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D002848:Chromatography, DEAE-Cellulose; D027101:DNA Topoisomerase IV; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D012689:Sequence Homology, Nucleic Acid
D004269:DNA, Bacterial; D027101:DNA Topoisomerase IV; D004264:DNA Topoisomerases, Type I