Thymidine metabolism in Escherichia coli K-12 : biochemistry of DNA labeling and the dynamics fo deoxyribonucleotide pools

Abstract

The DNA labeling characteristics of several E. coli K-12 strains were investigated under various labeling conditions. These strains differed in their thymine requirements (Thy), their ability to actively take up thymidine (Nup), and their ability to degrade thymidine (Tpp). In support of these studies on DNA labeling, the dynamics of the deoxyribonucleotide pools, particularly the deoxythymidine nucleotide pools, were examined using HPLC. The pool studies indicate that exogeneously supplied labeled thymine or thymidine is channeled directly into the DNA without equilibration with the existing pools in both Thy('+) and Thy('-) strains. These studies also show that the incorporation of label is more dependent on the rate of uptake than on the rate of passage through the intervening metabolic steps. From the comparisons made the incorporation studies and the information on the pool dynamics the following recommendations for labeling DNA are made: (1) For all short labeling periods a Thy('+) strain should be used and labeled with thymidine or dTMP. (2) For all long-term labeling (one-half of a generation time or longer) a Thy('-) strain should be used and labeled with thymine. (3) All other exogenously supplied nucleosides should be avoided wherever possible.