ROLE OF RIBONUCLEOTIDE REDUCTASE AND DEOXYNUCLEOTIDE POOLS IN THE OXYGEN-DEPENDENT CONTROL OF DNA-REPLICATION IN EHRLICH ASCITES-CELLS

Cultured Ehrlich ascites cells were exposed to different oxygen tensio ns (ranging from nearly complete anoxia to 95% O-2 at 10(5) Pa) and to transient (5-10 h) hypoxia (0.02% O-2 at 10(5) Pa). Treated cells wer e examined with respect to the intracellular concentration of the M2-s pecific tyrosyl free radical of ribonucleotide reductase by EPR spectr oscopy, and with respect to the pool sizes of all four deoxynucleoside triphosphates by an enzymatic assay employing DNA polymerase I of Esc herichia coli. From 2% to 0.02% O-2, the free radical level decreased continually from a normal value to just above detectability by the EPR measurement employed, and quickly recovered when hypoxic cells were r esupplied with atmospheric O-2. Concurrently, analogous changes of the size of the dCTP pool occurred, whereas the pool sizes dATP and dGTP underwent no changes, and the size of the dTTP pool only moderate chan ges. The changes of the free radical concentration and of the dCTP poo l correlated well with the suppression or reactivation of DNA replicat ion under the respective O-2 conditions. The results consistently supp ort the hypothesis of a fast-acting regulatory pathway that controls t he rate of DNA replication in proliferating cells according to suffici ent availability of O-2. Therefore, ribonucleotide reductase may serve , in addition to providing DNA building blocks, as a pO(2) sensor, whi ch transmits the signal in the form of an altered intracellular dCTP c oncentration, directly or indirectly, to the nuclear-replication machi nery.