• Identification of proteins that interact with Escherichia coli DNA topoisomerase III in vivo

      Kumar, Uplaksh S.; DiGate, Russell J. (2000)
      Topoisomerase III (Topo III) belongs to the type I DNA Topoisomerase family and is encoded by the topB gene. This enzyme is capable of relaxing and decatenating DNA. Although structure-activity relationships within the protein have been extensively studied, there is very limited information about the function of the enzyme in vivo. In this dissertation we have been able to identify proteins that interact with Topo III based on phage display library and biochemical purification. A region of Penicillin Binding Protein 1B (PBP1B) of E. coli was identified by phage display. This enzyme catalyzes the polymerization and insertion of peptidoglycan precursors into the bacterial cell wall during cell elongation. Using a modified Elisa assay we have confirmed that PBP1B and Topo III interact in vitro. To study the in vivo interaction, a gfp-topB (green fluorescence protein) fusion protein was constructed and the localization of Topo III in a PBP1B deletion mutant and its isogenic parent strain was observed under fluorescence microscopy. Topo III is located at the septum and the poles in the wild type cells, however no localization of Topo III was found in PBP1B mutant cells. In addition, a lethal effect due to the overexpression of Topo III can be rescued in a PBP1B mutant. All these results indicate that the two proteins interact in vivo. The localization of a mutant Topo III has also been examined. The overexpression Topo III containing a G432D mutation is not lethal to the cells. No localization of this mutant was observed regardless of the presence or absence of PBP1B. This suggests that the mutation may identify the region of Topo III that interacts with PBP1B. A biochemical approach has been designed to identify additional proteins that interact with Topo III. A partially purified protein has been shown to inhibit the relaxation activity of Topo III. This protein may be a useful candidate in the design of specific inhibitors of Topo III. In conclusion, the function of Topo III is not only restricted to DNA metabolism but also plays a role in cell division. The identification of the inhibitor of Topo III could also yield a new antibiotic.
    • Structural basis of Escherichia coli DNA topoisomerase III-catalyzed decatenation

      Li, Zhiyu; DiGate, Russell J. (1999)
      Escherichia 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.
    • Structure/function analysis of Escherichia coli DNA topoisomerase III

      Zhang, HongLiang; DiGate, Russell J. (1994)
      The generalized nucleic acid-binding domain of Escherichia coli DNA topoisomerase III (Topo III) has been identified using a selection procedure designated to isolate inactive Topo III polypeptides. Deletion of this binding domain, contained within the carboxyl terminal 50 amino acid residues of Topo III, results in a drastic reduction in the ability of the enzyme to bind to single-stranded DNA and RNA substrates. Successive truncation of the enzyme within this region results in the gradual loss of nucleic binding activity and in a gradual change in the mechanism of Topo III-catalyzed relaxation of negatively supercoiled DNA. Truncation of the enzyme to 605 amino acids (Topo III contains 653 amino acids) reduced DNA relaxation activity and binding activity to 1-2% the activity of the full length polypeptide. The reduction of nucleic acid binding activity of the truncated polypeptides does not result in a loss of cleavage site specificity for the enzyme, suggesting other amino acids are involved in the positioning of the nucleic acid within the nicking/closing site of the topoisomerase. Topo III binds asymmetrically to its substrate relative to its cleavage site. Topo III protects 14 nucleotides (12 nucleotides upstream of the cleavage site and 2 nucleotides downstream) from nuclease P1 digestion. The minimum substrate requirement for Topo III-catalyzed cleavage of DNA is only 7 nucleotides; however, this minimum substrate exhibits the same asymmetric sequence preference (6 nucleotides upstream of the cleavage site and 1 nucleotide downstream). In addition, a substrate that contained a Topo III cleavage/binding site formed a more stable complex with the enzyme than a substrate that did not contain a Topo III cleavage/binding site. An oligonucleotide with a Topo III cleavage/binding site was a better competitor for Topo III than an oligonucleotide without the site. Based on these results, a model is proposed in which Topo III binds to DNA and diffuses along the substrate until a cleavage site is located. When a cleavage site is found, Topo III halts and stably binds to the cleavage site. A hybrid of molecule consisting of Topo III cleavage domain and the Topo I binding domain has been used to demonstrate that the domains for cleavage and binding could be separated. The hybrid was created by fusing the first 604 amino acids of Topo III with the carboxyl-terminal 313 amino acids of Topo I. Addition of the C-terminal amino acids of Topo I restored both DNA relaxation activity and DNA binding activity to the enzyme; however, the ability of the enzyme to segregate newly synthesized replication molecules was not drastically enhanced. This suggests that C-terminal domain of Topo III may specifically contribute to its ability to decatenate newly replicated chromosomes.