Bacillus cereus DNA topoisomerase I and IIIalpha: purification, characterization and complementation of Escherichia coli TopoIII activity.

The Bacillus cereus genome possesses three type IA topoisomerase genes. These genes, encoding DNA topoisomerase I and IIIalpha (bcTopo I, bcTopo IIIalpha), have been cloned into T7 RNA polymerase-regulated plasmid expression vectors and the enzymes have been overexpressed, purified and characterized. The proteins exhibit similar biochemical activity to their Escherichia coli counterparts, DNA topoisomerase I and III (ecTopo I, ecTopo III). bcTopo I is capable of efficiently relaxing negatively supercoiled DNA in the presence of Mg2+ but does not possess an efficient DNA decatenation activity. bcTopo IIIalpha is an active topoisomerase that is capable of relaxing supercoiled DNA at a broad range of Mg2+ concentrations; however, its DNA relaxation activity is not as efficient as that of bcTopo I. In addition, bcTopo III is a potent DNA decatenase that resolves oriC-based plasmid replication intermediates in vitro. Interestingly, bcTopo I and bcTopo IIIalpha are both able to compensate for the loss of ecTopo III in E.coli cells that lack ecTopo I. In contrast, ecTopo I cannot substitute for ecTopo III under these conditions.