Abstract
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.
Highlights
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
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
An earlier study had shown that the combined rates of the de nouo syntheses of HmdCMP
Summary
HmCyt, 5-hydroxymethylcytosine; HmdCMP. 5-hvdroxvmethvl dCMP; Dna-, DNA-svnthesis negative (this designatibn, which is” in keeping with bacterial genetic tkrminoiogy, has been used rather than DO, generally employed for phage T4). Since the deoxyribonucleotides reach high levels under these circumstances, it was proposed that feedback effector controls might not play significant roles in their regulation [4, 9]. This suggestion was made even though several of the enzymes in the deoxyribonucleotide synthesis pathways are known to respond to positive [11,12,13] or negative [13] effector action or to product inhibition (14--16). The apparent lack of feedback control even with the high levels of nucleotides accumulating after infection by Dna- mutants [4, 17, 18], led us to ask whether deoxyribonucleotides are intrinsically synthesized at the ratio of (Ade + Thy)/(Gua. The present study gives evidence that, the ratio of thymine and 5hydroxymethylcytosine deoxyribonucleotides synthesized de novo is accurately regulated at 2:1, and that this precise regulation is primarily the inherent property of the several constituent enzyme systems
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