This research uses T4 bacteriophage for explorations of relationships between DNA precursor biosynthesis and DNA replication. The intracellular organization and control of the enzymes of deoxyribonucleoside triphosphate (dNTP) production affects both rate and fidelity of DNA replication. Since many anticancer, antiviral, and antimicrobial drugs act via interference with dNTP biosynthesis, further understanding of these relationships may suggest more effective ways to use existing drugs. T4 phage-infected bacteria form a complex of viral and host enzymes of dNTP synthesis, which seems to be juxtaposed with the replication apparatus and maintains local DNA precursor concentrations at the high levels needed to sustain vigorous DNA replication. This research will continue to test the model described above. This involves investigations of the structure, activities, assembly, and regulation of the isolated T4 dNTP-synthesizing complex, and purification of most or all of the constituent enzymes in quantities sufficient for attempted reconstitution studies and for analysis of protein-protein and protein-nucleic acid interactions. These experiments will involve affinity chromatography using immobilized protein columns, fluorescence anisotropy studies, and extension of experiments we have begun with protein cross-linking agents. Other projects will investigate parameters of dNTP biosynthesis and compartmentation as possible determinants of the fidelity of DNA replication. This will involve in vivo studies, where genetic lesions affecting DNA precursor synthesis are correlated with dNTP pool sizes and both spontaneous and analog-induced mutation rates; and in vitro studies, which determine the effects of high dNTP concentrations upon replication error frequencies at defined loci. Finally, investigations of gene organization and expression in the region of the T4 genome between genes 63 and 32 will continue. Sequence determination of gene frd, which encodes dihydrofolate reductase (DHFR), generated some surprises, including an overlap with the downstream td gene, which encodes thymidylate synthase, and an upstream open reading frame which in some respects looks like a DHFR pseudogene. The biological significance of these features will be studied.
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