Duan D S, Sadee W
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143.
Biochem J. 1988 Nov 1;255(3):1045-8. doi: 10.1042/bj2551045.
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 microM), 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.
研究了dGTP合成及其掺入DNA的两条替代途径：鸟嘌呤（Gua）→GMP→GDP→dGDP→dGTP→DNA和dG→dGMP→dGDP→dGTP→DNA。为了独立确定每条途径对DNA合成的贡献，在双突变S-49小鼠T淋巴瘤细胞系dGuo-L中进行了[14C]Gua和[3H]dG示踪实验，该细胞系缺乏嘌呤核苷磷酸化酶（EC 2.4.2.1）且具有dGTP反馈抗性核糖核苷酸还原酶（RR，EC 1.17.4.1）。在该细胞系中，外源性dG可选择性地提高dGTP池水平，而不影响RR和DNA合成。尽管在异步细胞中，[3H]dG（而非[14C]Gua，高达200 microM）能以剂量依赖性方式使细胞内dGTP池水平升高，但进入DNA的Gua通量中只有一小部分来自[3H]dG，大部分来自[14C]Gua。对G1期和部分富集的S期细胞进行的高效液相色谱分析表明，由于DNA合成过程中dGTP池的快速周转，[3H]dGTP仅在G1期细胞中积累，而在S期细胞中不积累。这些结果未能为细胞内dGTP的区室化提供证据，并表明单独的dG→dGMP→dGDP→dGTP途径维持DNA合成的能力不足。
