Changes of deoxyribonucleoside triphosphate pools induced by hydroxyurea and their relation to DNA synthesis.

V Bianchi,E Pontis,P Reichard

doi:10.1016/s0021-9258(18)66672-4

Abstract

Hydroxyurea inactivates ribonucleotide reductase from mammalian cells and thereby depletes them of the deoxynucleoside triphosphates required for DNA replication. In cultures of exponentially growing 3T6 cells, with 60-70% of the cells in S-phase, 3 mM hydroxyurea rapidly stopped ribonucleotide reduction and DNA synthesis (incorporation of labeled thymidine). The pool of deoxyadenosine triphosphate (dATP) decreased in size primarily, but also the pools of the triphosphates of deoxyguanosine and deoxycytidine (dCTP) were depleted. Paradoxically, the pool of thymidine triphosphate increased. After addition of hydroxyurea this pool was fed by a net influx and phosphorylation of deoxyuridine from the medium and by deamination of intracellular dCTP. An influx of deoxycytidine from the medium contributed to the maintenance of intracellular dCTP. 10 min after addition of hydroxyurea, DNA synthesis appeared to be completely blocked even though the dATP pool was only moderately decreased. As possible explanations for this discrepancy, we discuss compartmentation of pools and/or vulnerability of newly formed DNA strands to nuclease action and pyrophosphorolysis.

Highlights

From mammalian cells and thereby depletes them of We found recently that hydroxyurea in 3T6 cells stimulated the deoxynucleoside triphosphates required for DNA the uptake of pyrimidine deoxyribonucleosidesfrom the mereplication
An influx of deoxycytidine from the medium contributed to the maintenance of intracellular dCTP. 10 min after addition of hydroxyurea, DNA synthesis appeared tobecomnucleoside contributed to the rise of the dTTP pool
How are thechanges in pool sizelinked to theinhibition of DNA synthesis? How rapidly does hydroxyurea inhibit ribonucleotide reduction and DNA synthesis? Are the kinetics of pool changes related to the inhibition of DNA synthesis? In this article we describe experiments with rapidly growing 3T6 cells that address these questions by analyzing short term effects of hydroxyurea on ribonucleotide reduction, dNTP

Summary

Introduction

From mammalian cells and thereby depletes them of We found recently that hydroxyurea in 3T6 cells stimulated the deoxynucleoside triphosphates required for DNA the uptake of pyrimidine deoxyribonucleosidesfrom the mereplication. From the average specific activity of the dCTPpool (650 cpm/pmol), we could calculate that during the 10 min 71 pmol of dCTP were lost from the pool, that 25 pmol were incorporated into DNA, and that 40 pmol were transformed into dTTP.

Results

Conclusion