Guanine for DNA synthesis. A compulsory route through ribonucleotide reductase

Two alternative pathways for the synthesis of dGTP and its incorporation into DNA were studied: guanine (Gua) → GMP → GDP → dGDP → dGTP → DNA and dG → dGMP → dGDP → dGTP → DNA. To determine the contribution of each pathway to DNA synthesis independently of each other, [¹⁴C]Gua and [³H]dG tracer experiments were performed in a double-mutant S-49 mouse T-lymphoma cell line, dGuo-L, with purine nucleoside phosphorylase (EC 2.4.2.1)-deficiency and dGTP-feedback-resistant ribonucleotide reductase (RR, EC 1.17.4.1). In this cell line, dGTP pools can be selectively elevated by exogenous dG without affect RR and DNA synthesis. Although [³H]dG, but not [¹⁴C]Gua (up to 200 μM), readily expanded the cellular dGTP pool in a dose-dependent fashion in asynchronous cells, only a small fraction of the Gua flux into DNA was derived from [³H]dG, with the major fraction coming from [¹⁴C]Gua. H.p.l.c. analysis of G₁- and partially enriched S-phase cells revealed that [³H]dGTP only accumulates in G₁- but not in S-phase cells because of a rapid turnover of the dGTP pool during DNA synthesis. These results fail to provide evidence for cellular dGTP compartmentation and suggest that the pathway dG → dGMP → dGDP → dGTP alone has insufficient capacity to maintain DNA synthesis.