Acid-Soluble Deoxynucleotides and DNA Synthesis in Growing Yeast after X-Irradiation, II. Synthesis of Deoxynucleoside Tri- and Monophosphates in Synchronized and Asynchronously Growing Cells

Abstract

The behaviour of acid-soluble DNA precursors in synchronized and asynchronously growing yeast after X-irradiation is investigated by labeling techniques with 32Pi and by enzymic estimation. In prelabeled synchronized growing cells, radioactivity associated with deoxynucleoside triphosphates increases to maximum values shortly before each DNA replication, followed by a drastic decrease during S-phase. Radioactivity associated with monophosphates fluctuates, too, but with an opposite rhythm. These fluctuations apparently reflect quantitative changes of the DNA precursor pool during a cell cycle, as judged from the following findings: 1. Acid-soluble phosphorus is augmented stepwise. 2. "Specific" radioactivity from acid-soluble phosphorus compounds decreases steadily, indicating a continuous dilution of the labeled phosphorus pool with "cold" phosphorus. 3. Radioactivity associated with ribonucleotides fluctuates, too, but with a divergent rhythm. In X-irradiated synchronice growing yeast, the fluctuations of the deoxynucleotide-associated 32P are disturbed only little. Maximum values appear nearly at the same time as in the control, they decrease to minor values even if DNA augmentation is delayed. This decrease is less drastic, however, than that during DNA replication in unirradiated yeast, yielding a slightly increased aver­ age label per generation time. At the same sime a rapid augmentation of monophosphate label is observed. A pronounced increase of deoxynucleotide-32P is seen with X-irradiated asynchronously growing yeast, pointing to distinct radiosensitivities of the DNA/DNA precursor system in different cell stages. Neither 32P-fluctuations nor 32P-accumulation during DNA delay can be explained by cor­ responding observations with acid-soluble phosphorus or with ribonucleotide pools. Studies on 32P-incorporation also exclude radiation effects on cellular phosphorus uptake. Enzymic estimations of the deoxynucleoside triphosphate pools from asynchronously growing yeast rather exhibit a considerable increase of these substances during the radiation-induced delay of DNA augmentation. This accumulation of DNA precursors probably is caused by undisturbed synthesis, but reduced incorporation into DNA. The possible role of DNA repair in this system is discussed.