From experimentally induced changes in the slope (Vmax) and intercept (pool) of isotope-dilution plots inferences may be drawn on the position and regulation of rate-limiting steps affecting the incorporation of pyrimidine deoxynucleosides by intact cells. 5-Fluorodeoxyuridine (FdUrd; 1 μM) reduced the Vmax of radioactive labelling with deoxy[5-3H]cytidine; this was reversed by thymidine (19 μM) suggesting that FdUrd makes the concentration of deoxythymidine triphosphate (dTTP) rate-limiting for DNA synthesis. With deoxy[U-14C]cytidine the reversal of FdUrd inhibition by thymidine was only partial; this was in keeping with (i) deoxy[U-14C]cytidine labelling both cytosine and thymine in DNA, and (ii) a continuing inhibition of thymidylate synthetase by FdUrd in the presence of thymidine (19 μM).The deoxycytidine competitor pool was increased by cytidine (10–50 μM) and decreased by (i) thymidine (19 μM), (ii) hydroxyurea (50 μM) and (iii) deoxycytidine (12 μM, in the presence of FdUrd). It is suggested that these pool-decreasing agents, or their derivatives (e.g., dTTP), inhibit ribonucleotide reductase and hence prevent the entry of pyrimidine ribonucleotide derivatives into the deoxycytidine competitor pool; because of this pool decrease, radioactive labelling with deoxy[5-3H]cytidine was enhanced by thymidine (19 μM) and hydroxyurea (50 μM). However, at the latter hydroxyurea concentration, labelling with [Me-3H]thymidine was inhibited, due to a decrease in the Vmax of the rate-limiting step for thymidine incorporation (probably thymidine kinase). This sensitivity of labelling with [Me-3H]thymidine to inhibition by hydroxyurea (50 μM) was reduced by adding FdUrd to prevent the accumulation of dTTP. At high hydroxyurea concentrations (0.1–1.0 mM), labelling with deoxy[5-3H]cytidine was also inhibited, due to a decrease in Vmax of the rate-limiting step, which was probably at the level of DNA polymerase.The results suggest that hydroxyurea inhibits DNA synthesis by making the concentration of purine deoxynucleotides rate-limiting. Pyrimidine deoxynucleotides are formed in sufficient quantities from deoxycytidine by way of salvage pathways. Indeed, dTTP accumulates and inhibits thymidine kinase, thus amplifying the inhibitory effect of hydroxyurea on labelling with [Me-3 H]thymidine.
The rate-limiting step of DNA synthesis by DNA polymerase occurs in the fingers-closed conformation
