T4 Phage dNTP Synthetase: A Multienzyme Complex for Deoxyribonucleotide Synthesis

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This research involves a multienzyme complex, formed in T4 phage- infected Escherichia coli, which carries out the synthesis of deoxyribonucleoside tripgisohates (dNTPs). This T4 dNTP synthesis complex will be investigated by both biochemical and genetic means, with the goals of learning how it functions and how it integrates dNTP synthesis with DNA replication. First, through analysis of complexes isolated from T4-infected cells and through partial reconstitution of the complex from purified proteins, we hope to define the protein composition, stoichiometry, and protein-protein interactions within the complex. Second, through assays for multi-step reaction pathways catalayzed by the purified complex, and through study of the allosteric properties of its constituent enzymes, we hope to learn how regulation of individual enzymes contributes toward control of the complex, and in turn helps to explain how dNTP synthesis is controlled in vitro. Third, through enzyme kinetic analysis, studies with bifunctional protein crosslinkers, and investigations of dNTP pool dynamics and DNA synthesis in vitro, we hope to learn whether the dNTP synthetase complex is physically or functionally linked to the DNA replication apparatus in vivo. Fourth, through reversion analysis at defined loci in the T4 rII cistrons, we will investigate mechanisms of mutagenesis induced by DNA precursor pool imbalances, with some of the data possibly generating insight into the nature of the microenvironment at DNA replication sites.