Browsing Theses and Dissertations School of Pharmacy by Title "Structural basis of Escherichia coli DNA topoisomerase III-catalyzed decatenation"
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Structural basis of Escherichia coli DNA topoisomerase III-catalyzed decatenationEscherichia coli topoisomerase III (Topo III) consists of four domains folding into a torus with a central cavity of approximately 25 A in diameter. In order to assess the DNA binding function of this hole, a pair of cysteine residues were introduced into Topo III at I299 and A494 in two adjacent domains near the hole. In the absence of reducing reagent, the C299 and C494 form a disulfide bond. By regulating the oxidation and reduction of this disulfide bond, a double stranded or a single stranded DNA has been trapped within the Topo III central hole. Topo III possesses a single stranded DNA binding region in addition to the central hole. A single stranded DNA gel shift assay has been applied to the Topo III/double stranded DNA complex, a slowly migrating complex consisting of the Topo III, double stranded DNA, and single stranded DNA has been observed. Based on these observations and other evidence, a 'strand passage' model of Topo III-catalyzed decatenation has been developed. A seventeen amino acid residue domain has been also identified in Topo III that is essential for Topo III-mediated resolution of DNA replication intermediates in vitro. Deletion of this domain completely abrogates Topo III-catalyzed resolution of DNA replication intermediates and decatenation of multiply linked plasmid DNA dimers. The modified polypeptide, however, was capable of relaxing negatively supercoiled DNA substrates. The presence of this domain has been detected in multiple plasmid encoded topoisomerases raising the possibility that these enzymes may also be decatenases. The polypeptide encoded by the plasmid RN traE gene shows extensive protein sequence homology with E. coli topB, the gene encoding Topo III. The traE gene product has been cloned, expressed and purified. The TraE protein exhibits topoisomerase activity similar to that of Topo III. Similar to E. coli Topo III, the TraE protein is a potent decatenase and can substitute for Topo III activity in vivo. The biochemical properties of the TraE protein in vitro suggest that the protein may be involved in the resolution of plasmid DNA replication intermediates during either vegetative replication, or in conjugative DNA transfer. Putative homologues of Topo III have been found to be encoded by other broad host range, conjugative plasmids isolated from both gram negative and gram positive organisms suggesting that Topo III-like polypeptides may have an essential role in the propagation of many promiscuous plasmids.