DIFFERENTIAL-EFFECTS OF HYDROXYUREA UPON DEOXYRIBONUCLEOSIDE TRIPHOSPHATE POOLS, ANALYZED WITH VACCINIA VIRUS RIBONUCLEOTIDE REDUCTASE

Hydroxyurea inhibits DNA synthesis by destroying the catalytically ess ential free radical of class I ribonucleoside diphosphate (rNDP) reduc tase, thereby blocking the de novo synthesis of deoxyribonucleotides. In mammalian cells, including those infected by vaccinia virus, hydrox yurea treatment causes a differential depletion of the four deoxyribon ucleoside triphosphate pools, suggesting that the activities of rNDP r eductase are differentially sensitive to hydroxyurea, In the presence of different substrates and allosteric modifiers, we measured rates of free radical destruction in the vaccinia virus-coded rNDP reductase, by following absorbance at 417 nm as a function of time after hydroxyu rea addition. Also, we followed enzyme activity directly, by using a r ecently developed assay that allows simultaneous monitoring of the fou r activities, in the presence of substrates and effecters at concentra tions that approximate the intracellular environment. We found the pri mary determinant of radical loss to be not the ensemble of allosteric ligands bound but the activity of the enzyme. Nucleoside triphosphate effecters accelerated radical decay, compared with rates seen with the free enzyme. Adding substrate to the holoenzyme, under conditions whe re the enzymatic reaction is proceeding, further accelerated radical d ecay, Alternative models are discussed, to account for selective deple tion of purine nucleotide pools by hydroxyurea.