Graduate Thesis Or Dissertation

Regulation of mouse ribonucleotide reductase by allosteric effector-substrate interplay and hypoxia

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  • In order to maintain genetic stability in eukaryotes, tight regulation of the relative sizes of deoxyribonucleoside triphosphate (dNTP) levels inside the cell is essential for optimal fidelity of DNA replication. Ribonucleotide reductase (RNR) is the enzyme responsible for proportional production of DNA precursors. Studies on regulation of this enzyme, the focus of this thesis, are important because mutations affecting RNR control mechanisms result in dNTP pool imbalance, thus promoting mutagenesis. By using mouse RNR as a model for mammalian forms of the enzyme, three major factors--allosteric effectors, rNDP substrate concentrations, and hypoxic conditions--that influence the substrate specificity of RNR have been investigated. Allosteric regulation has been studied by the four-substrate assay, which permits simultaneous monitoring of the four reactions catalyzed by this enzyme in one reaction mixture. Individual dNTPs affect the four activities differentially in a concentration-dependent manner with discrete effects of dTTP and dGTP on reduction of ADP and GDP, respectively. Ribonucleoside diphosphate (rNDP) substrate concentrations are equally important, as their variations lead to different product ratios. Results from nucleotide binding assays indicate that rNDPs directly influence binding of dNTP effectors at the specificity site, one of the two classes of allosteric sites, whereas ADP has an indirect effect, displacing other substrates at the catalytic site and consequently removing effects of those substrates upon dNTP binding. Hence, this is the first evidence of a two-way communication between the catalytic site and the specificity site. Oxygen limitation also plays an important role in controlling the enzyme specificity. Reactivation of the enzyme at different oxygen tensions, after treatment of the enzyme with hydroxyurea (HU) followed by removal of HU, reveals a distinct sensitivity of GDP reductase to low 0₂ levels. Although the basis for specific inhibition of GDP reduction remains to be determined, some possibilities have been ruled out. This research proves that in addition to allosteric regulation by nucleoside triphosphates, mouse RNR is also controlled by other factors. Since these components can simultaneously exert their effects upon enzyme specificity, complex regulatory patterns of RNR to provide a proportional supply of the DNA building blocks in vivo are suggested.
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