Browsing Theses and Dissertations School of Pharmacy by Title "Identification of proteins that interact with Escherichia coli DNA topoisomerase III in vivo"
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Identification of proteins that interact with Escherichia coli DNA topoisomerase III in vivoTopoisomerase 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.