Escherichia coli DNA topoisomerase III: purification and characterization of a new type I enzyme.
A new topoisomerase capable of relaxing negatively supercoiled DNA in Escherichia coli has been identified during chromatography on novobiocin-Sepharose. A simple and reproducible purification procedure is described to obtain this enzyme, called topoisomerase III (topo III), in a homogeneous form. The protein is a single polypeptide with a molecular weight of 74 000 +/- 2000 and is a type I topoisomerase, changing the linking number of DNA circles in steps of one. It is present in deletion strains lacking the topA gene and further differs from the well-studied topoisomerase I (omega protein; Eco topo I) in (1) its requirement for K+ in addition to Mg2+ to exhibit optimal activity and (2) its affinity to novobiocin-Sepharose. Positively supercoiled DNA is not relaxed during exposure to the enzyme. Topo III has no ATPase activity, and ATP does not show any discernible effect on the reduction of superhelical turns. The purified topoisomerase has no supercoiling activity and is unaffected by high concentrations of oxolinic acid and novobiocin in the relaxing reaction. Single-stranded DNA and spermidine strongly inhibit the topoisomerase activity.
D002846:Chromatography, Affinity; D004247:DNA; D004264:DNA Topoisomerases, Type I / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D009675:Novobiocin; D011188:Potassium / Q000494:pharmacology
D009675:Novobiocin; D004247:DNA; D004264:DNA Topoisomerases, Type I; D008274:Magnesium; D011188:Potassium