Ribonucleotide Reductase and DNA Precursor Pool Control

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Ribonucleotide reductase reduces four ribonucleotide substrates to the corresponding 2'-deoxyribo-nucleotides in the first reaction committed to DNA synthesis. The enzyme is regulated by nucleoside triphosphate effectors that interact with two classes of allosteric sites to control the specificity of the enzyme, so that deoxyribonucleoside triphosphates (dNTPs) are synthesized in proportion to the base composition of the genome. Using an assay procedure that monitors the four reactions in one mixture, at substrate and effector concentrations similar to those in vivo, the project will investigate several interrelated aspects of the control of dNTP pool sizes in mammalian cells. Because balanced dNTP pools are essential for accurate DNA replication, these studies should illuminate how cells determine the fidelity as well as the rate of DNA replication.1. ADP has been found to specifically inhibit the reduction of GDP and of ADP itself. Using the four-substrate assay, competitive binding experiments, and photoaffinity labeling, this project will ask whether the effect involves ADP binding at the catalytic site, an allosteric site, or a previously undescribed site.2. Hydroxyurea, a free radical scavenger, inhibits all four activities of rNDP reductase. However, treating cells with hydroxyurea causes preferential depletion of the dATP pool. This project will ask whether this effect results from accumulation of ADP, with consequent inhibition of ADP reduction in vivo. An alternative model relates to the selective depletion of dGTP, the prime allosteric activator of ADP reduction.3. Culturing cells under hypoxic conditions has been found to selectively shrink the dCTP pool, while the other three dNTPs accumulate. Since oxygen is required for rNDP reductase activation, there may be a specific inhibition of the activation of CDP reduction at low pO2. This hypothesis will be tested by use of the four-substrate assay mentioned above.4. dNTPs within mitochondria comprise pools distinct from the larger pools that serve the nuclear genome. Also, mitochondria contain a distinctive rNDP reductase. The mitochondrial enzyme will be purified and characterized with regard to regulation, genetic origin, and role in synthesizing mtDNA precursors.This research is designed to help us understand how enzymes function in their natural environment, the living cell, using as the principal enzyme ribonucleotide reductase, which catalyzes the first reaction committed to DNA synthesis. Possibly, significant insights will be gained into relationships between energy metabolism and genome replication, into the control of DNA replication, into cellular responses to oxygen stress, and into mitochondrial DNA replication.