The effects of different thymidine concentrations on DNA replication in pea-root cells synchronized by a protracted 5-fluorodeoxyuridine treatment

Abstract

Single-cell and DNA fiber autoradiography, cytophotometry and velocity sedimentation in alkaline sucrose gradients were used to analyse DNA replication and nascent replicon maturation in 5-fluorodeoxyuridine (FUdR)-synchronized cells of Pisum sativum. The replicon size was not significantly changed by the protracted FUdR treatment. When the synchronized cells were released from the inhibitor, labeled with [ 3H]TdR for 30 min, and chased in medium containing 1 × 10 −6 M or lower concentrations of cold thymidine, DNA replication stopped after approx. 25% of the genome had replicated, and the nascent strands failed to grow above 9–12 × 10 6 D single-stranded (ss) DNA. When the cells were chased in medium with 1 × 10 −5 M cold thymidine, the DNA content of the labeled cells steadily increased with time and the size of the nascent molecules grew continuously until replicon size was achieved; then they were accumulated at replicon size until the cells arrived in late S or G2. When the FUdR-synchronized cells were chased in medium containing 1 × 10 −4 M cold thymidine, the size of the nascent strands increased continuously with time, indicating that some neighbouring nascent replicons were joined as soon as they completed their replication. These observations led us to postulate that in FUdR-synchronized cells the rates of chain elongation, cell progression through the S phase and nascent replicon maturation are controlled by thymidine availability.