The kinetics of the de novo formation of
pyrimidine deoxyribonucleotides is the same after
infection by wild type bacteriophage T4, which generate very low steady state levels of deoxytibonucleotides, and by T4
DNA synthesis-negative mutatants (
Dna-), which accumulate high levels, suggesting that the control is not by a feedback mechanism. In this study, the ratio of the de novo synthesis of
dTMP to HmdCMP derivatives was measured by determining the total
thymine and 5-hydroxylxytosine (HmCyt)
deoxyribonucleotides synthesized by the reductive pathways from [6-3H]
uracil including those in
DNA and any degradation products excreted into the medium. The ratio of the de novo 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
Dna-genes 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 100-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
Dna- mutants tested excreted 35 to 50% of their
thymine derivatives, mostly as
thymine, into the medium. Neither
thymine nor
5-hydroxymethyluracil derivates were excreted after wild type phage
infection. We propose that
pyrimidine deoxyribonucleotide synthesis is regulated at a Thy:HmCyt ratio of 2:1 as an intrinsic property of a complex of
enzymes synthesizing and channeling
deoxyribonucleotides for T4 DNA replication and not exclusively by effector-sensitive mechanisms.