Regulation of Deoxyribonucleotide Biosynthesis during in Viva Bacteriophage T4 DNA Replication INTRINSIC CONTROL OF SYNTHESIS OF THYMINE AND 5HYDROXYMETHYLCYTOSINE DEOXYRIBONUCLEOTIDES AT PRECISE RATIO FOUND IN DNA*

The kinetics of the de nouo formation of pyrimidine deoxyribonucleotides is the same after infection by wild type bacteriophage T4, which generate very low steady state levels of deoxyribonucleotides, and by T4 DNA synthesis-negative mutants (Dna-1, which accumulate high levels, suggesting that the control is not by a feedback mechanism. In this study, the ratio of the de nouo synthesis of dTMP to HmdCMP derivatives was measured by determining the total thymine and Shydroxymethylcytosine (HmCyt) deoxyribonucleotides synthesized by the reductive pathway from [6“Hluracil including those in DNA and any degradation products excreted into the medium. The ratio of the de nouo synthesis of Thy/HmCyt derivatives remained constant at 2.1 + 0.1 for at least 45 min after infection by wild type phage, i.e. precisely at the Thy/ HmCyt ratio in T4 DNA. On infection by phage mutated in the Dnagenes 32,41,44, or 45, the ratio still remained close to 2 to 1 for at least 25 min. Only after the pyrimidine deoxyribonucleotide concentrations reached levels about loo-fold greater than the initial values did the ratio begin to increase. However, a mutant of the structural gene for T4 DNA polymerase showed some increase in ratio by 15 min. Mutants of gene 1 (HmdCMP kinase) were distinct in that the Thy/HmCyt ratio dropped to about 1.0 by 25 min, and then remained quite constant. Uniquely, in these mutants a significant quantity of 5-hydroxymethyluracil or a derivative was found, about 40% being in the medium. The product was shown to be derived by deamination of a 5-HmCyt derivative. All Dnamutants tested excreted 35 to 50% of their thymine derivatives, mostly as thymine, into the medium. Neither thymine nor 5-hydroxymethyluracil derivatives were excreted after wild type phage infection. We propose that pyrimidine deoxyribonucleotide synthe-