Integrase catalyzes insertion of a retroviral genome into the host chromosome. Following reverse transcription, integrase binds specifically to the ends of the duplex retroviral DNA, endonucleolytically cleaves two nucleotides from each 3'-end (the processing activity), and inserts these ends into the host DNA (the joining activity) in a concerted manner. Additionally, it has been observed that integrase can catalyze the removal of inserted viral ends (the disintegration activity) in vitro. Presteady-state experiments were performed using synapsed substrates to probe the processing reaction and a disintegration substrate to determine the number of protomers in a functional multimeric complex. In single-turnover studies, a novel "splicing" reaction was observed that revealed complications with accurate quantification of enzymatic activity using the synapsed substrates. The splicing reaction was further used to gain insight into the selection of nucleophiles and electrophiles at the binding site. To reduce the complexity introduced by the integrase-catalyzed splicing reaction, 5'-5' reverse-polarity synapsed substrates were designed that were not susceptible to the splicing reaction and that allowed direct comparison of LTR ends simultaneously bound at the active site. Analysis of the presteady-state assays using these reverse-polarity substrates revealed that the concurrent binding of the biologically relevant U3/U5 combination of viral ends facilitates maximal activity of the processing reaction. A disintegration substrate was used in presteady-state active site titrations to determine a reaction stoichiometry of four integrase protomers per one substrate molecule for the disintegration reaction. A tetrameric active complex was then confirmed using atomic force microscopy to image integrase-DNA complexes during the first catalytic turnover. The observed increase of the tetramer population in the presence of substrate DNA demonstrates that the binding of the disintegration substrate induces assembly of the active tetramer and suggests that tetramer assembly may be an integral and dynamic component of the catalytic pathway. / Graduation date: 2003

Bibliography: leaves 116-117 / viii, 131, [82] leaves, [20] leaves of plates : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, 1983

With respect to a multienzyme complex of deoxyribonucleoside triphosphate (dNTP) synthesis somehow juxtaposed with DNA replication sites, our laboratory has demonstrated the existence of a multienzyme complex in T4-infected E. coli, named the T4 dNTP synthetase complex, but the idea of direct linkage of dNTP synthesis to DNA replication and organization of the complex has not been well established. This study had two objectives. The first objective was to test the specific hypothesis that gp32, the single-stranded DNA binding protein encoded by gene 32, plays a role in recruiting enzymes of dNTP synthesis to the replisome and in organizing the dNTP synthetase complex. By use of two newly created gene 32 mutants along with several experimental approaches, DNA-cellulose chromatography, coimmunoprecipitation, and glutathione-S-transferase pull downs, interactions of gp32 with thymidylate synthase (gptd), ribonucleotide reductase (gpnrdA/B), and E. coli NDP kinase have been identified. These results support the hypothesis that gp32 helps to recruit enzymes of dNTP synthesis to DNA replication sites. As the second objective, I investigated contributions of two host proteins, E. coli nueleoside diphosphate kinase (NDP kinase) and adenylate kinase (Adk), to the organization of the complex. As an important step to understand roles of E. coli NDP kinase in the complex, I identified direct interactions of E. coli NDP kinase with gpnrdA/B, dCMP hydroxymethylase (gp42), and dihydrofolate reductase (gpfrd) by means of coimmunoprecipitation and glutathione-S-transferase pull-down experiments. Interestingly, these interactions were influenced by the presence of substrate nucleotides or an analog for E. coli NDP kinase, suggesting that metabolite flux may affect the preference of E. coli NDP kinase binding to enzymes in the complex in vivo. Meanwhile, Adk involvement in DNA precursor synthesis has been suggested, particularly in phage T4-infected E. coli, from observations of increased thermostability of temperature-sensitive Adk in situ. The involvement of E. coil Adk in the complex was demonstrated by identifying some proteins of the T4 dNTP synthetase complexgp42, dNMP kinase (gpl), gpfrd, and E. coli NDP kinasedirectly interacting with Adk, implying that E. coil Adk would be properly located in the complex to efficiently carry out the conversion of dNDPs to dNTPs. This implication was supported by measurements of T4 DNA synthesis. Taken together, this research strongly supports the idea of connection of dNTP synthesis to DNA replication and allows us to take a step toward understanding the organization of the complex at DNA replication sites. / Graduation date: 2005