| 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 nucleoside 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. coli Adk in the complex was demonstrated by identifying some proteins of the T4 dNTP synthetase complex---gp42, dNMP kinase (gp1), gpfrd, and E. coli NDP kinase---directly interacting with Adk, implying that E. coli 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. |
