Role of ribonucleotide reductase and deoxynucleotide pools in the oxygen-dependent control of DNA replication in Ehrlich ascites cells.

Abstract

Cultured Ehrlich ascites cells were exposed to different oxygen tensions (ranging from nearly complete anoxia to 95% O2 at 10(5) Pa) and to transient (5-10 h) hypoxia (0.02% O2 at 10(5) Pa). Treated cells were examined with respect to the intracellular concentration of the M2-specific tyrosyl free radical of ribonucleotide reductase by EPR spectroscopy, and with respect to the pool sizes of all four deoxynucleoside triphosphates by an enzymatic assay employing DNA polymerase I of Escherichia coli. From 2% to 0.02% O2, 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 resupplied with atmospheric O2. 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 changes. The changes of the free radical concentration and of the dCTP pool correlated well with the suppression or reactivation of DNA replication under the respective O2 conditions. The results consistently support the hypothesis of a fast-acting regulatory pathway that controls the rate of DNA replication in proliferating cells according to sufficient availability of O2. Therefore, ribonucleotide reductase may serve, in addition to providing DNA building blocks, as a pO2 sensor, which transmits the signal in the form of an altered intracellular dCTP concentration, directly or indirectly, to the nuclear-replication machinery.