While investigating the basis for marked natural asymmetries in deoxyribonucleoside
triphosphate (dNTP) pools in mammalian cells, we observed that culturing V79 hamster lung cells in a 2%
oxygen atmosphere causes 2-3-fold expansions of the dATP,
dGTP, and
dTTP pools, whereas
dCTP declines by a comparable amount. Others have made similar observations and have proposed that, because O(2) is required for formation of the catalytically essential
oxygen-bridged
iron center in
ribonucleotide reductase,
dCTP depletion at low
oxygen tension results from direct or indirect effects upon
ribonucleotide reductase. We have tested the hypothesis that
oxygen limitation affects
ribonucleotide specificity using recombinant mouse
ribonucleotide reductase and an assay that permits simultaneous monitoring of the reduction of all four
nucleotide substrates. Preincubation and assay of the
enzyme in an anaerobic chamber caused only partial activity loss. Accordingly, we treated the
enzyme with
hydroxyurea, followed by removal of the
hydroxyurea and exposure to atmospheres of varying
oxygen content. The activity was totally depleted by
hydroxyurea treatment and nearly fully regained by exposure to air. By the criterion of activities regained at different
oxygen tensions, we found
CDP reduction not to be specifically sensitive to
oxygen depletion; however,
GDP reduction was specifically sensitive. The basis for the differential response to reactivation by O(2) is not known, but it evidently does not involve varying rates of reactivation of different allosteric forms of the
enzyme or altered response to allosteric effectors at reduced
oxygen tension.