DNA PRECURSOR DYNAMICS IN CELLS

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Enzymes of DNA precursor biosynthesis are targets for the actions of anticancer, antiviral, and antimicrobial drugs, because the metabolic roles of deoxyribonucleoside triphosphates dNTPs) are limited to their functions as DNA precursors, inhibition of the synthesis of a particular deoxyribonucleotide presents a selective way to block DNA replication in a neoplastic cell or an infected cell. Effective use of nucleic acid antimetabolites in chemotherapy requires understanding of the intracellular organization and regulatory mechanisms affecting the enzymes of dNTP biosynthesis. Such understanding may lead also to insight into the control of DNA synthesis--both replication and repair. Specific aims of this program include: (1) to define mechanisms controlling the intracellular distribution of DNA precursor pools; (2) to define relationships between effective DNA precursor concentrations and the rate and accuracy of DNA replication; (3) to define intracellular associations among enzymes of dNTP biosynthesis; and (4) to evaluate the hypothesis that deoxyribonucleotide pools are significant targets for the mutagenic effects of alkylating agents. This project uses cultured mammalian cells and vaccine virus as biological systems for analyzing intracellular associations among dNTP biosynthetic enzymes and the intracellular distribution of DNA precursor pools. Sub- projects focus upon (1) the structure and regulation of a virus-coded form of ribonucleotide reductase, (2) deoxyribonucleotide pools as targets for spontaneous and chemically induced mutagenesis, (3) analysis of cell mutants with specific alterations in dNTP metabolism, (4) determination of the basis for sequestration of nucleotide pools in nuclei and in mitochondria, and (5) analysis of supramolecular assemblies involving ribonucleotide reductase and other enzymes of dNTP biosynthesis.