Oxygen dependent regulation of mammalian ribonucleotide reductase in vivo and possible significance for replicon initiation

Abstract

The intracellular concentration of the M2 specific free tyrosyl radical of ribonucleotide reductase in cultured Ehrlich ascites cells was estimated by EPR spectroscopy during imposition and after reversal of hypoxia. Under the same conditions, the intracellular intensity of CDP reduction was estimated indirectly by measuring the incorporation of radioactivity into DNA from labeled ribo- and deoxyribo-cytidine, respectively. The radical concentration distinctly decreased under hypoxia and reincreased upon reaeration. At the same time, the CDP reduction was greatly diminished and reactivated, respectively. These observations are interpreted in the sense of an O 2 dependent regulation of the intracellular activity of ribonucleotide reductase. The O 2 dependent deactivation and reactivation of the enzyme was temporally related to a specific suppression or a burst-like release of replicon initiations, respectively. Addition of 100 μM dCtd to hypoxic cells could substitute for reoxygenation with respect to triggering replicon initiations. A possible implication of the intracellular ribonucleotide reductase activity and the size of the dCTP pool in replicon initiation is dicussed.