V. Bianchi et al., CELL CYCLE-DEPENDENT METABOLISM OF PYRIMIDINE DEOXYNUCLEOSIDE TRIPHOSPHATES IN CEM CELLS, The Journal of biological chemistry, 272(26), 1997, pp. 16118-16124
We incorporated H-3-labeled thymidine, deoxycytidine, or cytidine into
dNTPs and DNA of exponentially growing CEM cells. G(1) and S phase ce
lls were separated by centrifugal elutriation, and the size and specif
ic activity of dNTP pools were determined to study the cell cycle-depe
ndent regulation of specific dNTP synthesizing enzymes in their metabo
lic context. With [H-3]thymidine, we confirm the earlier demonstrated
S phase specificity of thymidine kinase. Incorporation of radioactivit
y from [5-H-3]deoxycytidine into dCTP occurred almost exclusively in G
(1) cells, During S phase, de novo synthesis by ribonucleotide reducta
se was switched on, resulting in a 70-fold dilution of [H-3]dCTP, conf
irming that ribonucleotide reductase is an S phase-specific enzyme, wh
ereas deoxycytidine kinase is not, [5-H-3]Cytidine appeared in dCTP al
most to the same extent in G(1) as in S phase, despite the S phase spe
cificity of ribonucleotide reductase, During S phase, DNA replication
greatly increased the turnover of dCTP, requiring a corresponding incr
ease in ribonucleotide reductase activity. During G(1), the enzyme mai
ntained activity to provide dNTPs for DNA repair and mitochondrial DNA
synthesis, The poor incorporation of isotope from deoxycytidine into
DNA earlier led to the suggestion that the nucleoside is used only for
DNA repair (Xu, Y-Z., Peng, H., and Plunkett, W. (1995) J. Biol. Chem
. 270, 631-637). The poor phosphorylation of deoxycytidine in S phase
provides a better explanation.