Turnover of the Deoxyribonucleoside Triphosphates in Escherichia coli 15 T during Thymine Starvation

Abstract

Since hydroxyurea specifically inhibits the biosynthesis of deoxyribonucleotides in Escherichia coli, analysis of the deoxyribonucleoside triphosphate pools following hydroxyurea addition makes possible a determination of the turnover of these compounds in vivo. This method has been used in the present work to study the effect of thymine starvation of E. coli 15 T on the turnover of dGTP, dATP, and dCTP. From the results obtained the following conclusions may be made. In exponentially growing cultures DNA synthesis can account for the entire turnover of the deoxyribonucleoside triphosphates. Removal of thymine from the growth medium results in the appearance of catabolic reactions that degrade dATP and dCTP. During the first 20 min of thymine starvation the rates of degradation of dATP and dCTP are of the same magnitude as those accounted for by DNA polymerization during exponential growth. After 75 min, however, the decay rate of dCTP has increased 5‐fold while the rate of dATP decay remains constant. During the entire period of thymine starvation the rate of decay of dGTP is very low, i.e. one sixth of that observed during exponential growth. The end products of the catabolic reactions involved are to a large extent excreted into the growth medium as diphenylamine‐reacting material. By determining the rate of accumulation of the individual deoxyribonucleoside triphosphates following thymine deprivation, and adding these values to the decay rates, determined in the presence of hydroxyurea, it is possible to estimate the endogenous rate of synthesis of dGTP, dATP and dCTP under these conditions. The results show that removal of thymine from E. coli 15 T results in a significant reduction in the rate of dGTP synthesis, essentially no effect on the rate of dATP synthesis, and an increasing rate of dCTP synthesis reaching 6‐fold values after 75 min of starvation. Copyright © 1971, Wiley Blackwell. All rights reserved