Enzyme associations in T4 phage DNA precursor synthesis

Abstract

A direct approach is described to the question: Are enzymes of DNA precursor synthesis organized into a supramolecular structure? This approach involved sedimentation analysis of several T4 phage-coded early enzyme activities in crude lysates of infected Eschenichia coli. One-third to one-half of several activities tested-dCMP hydroxymethylase, dTMP synthetase, deoxynucleoside 5'-monophoso ate inase, deoxyuridine triphosphatase, and probably dCMP deaminase, but not dihydrofolate reductase or DNA polymerase-sedimented much more rapidly than expected from molecular weight. About 5% of the host cell nucleoside diphosphate kinase, known to participate in T4 DNA precursor synthesis, cosedimented with these activities. To show that this rapidly sedimenting material represents an organized enzyme complex rather than a nonspecific aggregate, we studied the kinetics of formation of dTTP with dUMP as the initial substrate. This three-step reaction sequence reached its maximal rate within a few seconds when catalyzed by enzymes in the aggregate, whereas an equivalent mixture of uncomplexed enzymes required nearly 20 Fig. 1 (reviewed in ref. 2). Certain observations point strongly to the existence of functional complexes involving deoxyribonucleotide-syn-The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.thesizing enzymes. First, cells infected with T4 mutants defective in gene 1 or 42, both of which code for DNA precursor enzymes, cannot synthesize DNA under in vitro conditions, even when the metabolic block is circumvented by provision of deoxynucleoside 5'-triphosphates (dNTPs) (3-6); second, enzyme activities can be detected in cell-free extracts even when the same enzymes are not functioning in the intact cell (7-9); third, a host cell enzyme, nucleoside diphosphate (NDP) kinase, is known to function in phage DNA precursor synthesis, yet studies on nucleotide pool kinetics suggest that only a small fraction of the total enzyme is available to function in this role (10); and fourth, the rates of deoxyribonucleotide synthesis in vivo seem to be regulated by the state of the viral DNA (11).Because the above approaches are largely indirect, we sought to demonstrate the existence of enzyme complexes by direct methods. We have confirmed the observation of Chiu and Greenberg (8) that much of the dCMP hydroxymethylase activity in a crude lysate of T4-infected cells is present in a rapidly sedimenting form, and several other activities behave similarly. This enzyme aggregate probably represents an organized complex because it efficiently catalyzes a multistep pathway in deoxynucleotide synthesis.MATERIALS AND METHODS Materials. Most experiments were carried out with phage T4D and E. coli B as host. Media included M9 and SM9 as described (6). Nucleotides, substrates, ...

Intracellular Location of Thymidylate Synthase and Its State of Phosphorylation

“…Recent studies indicate that this enzyme may be regulated at both the transcriptional (8) and translational levels of synthesis (9), while earlier studies suggested that TS forms multienzyme complexes involved in DNA synthesis both in T-even phage-infected E. coli (10,11) and eukaryotic cells (12,13). These findings are compounded further now by the enzyme's apparent association with the nucleolus of the cell and its state of phosphorylation, as will be described in this paper.…”

Bacteriophage T4 nrdA and nrdB genes, encoding ribonucleotide reductase, are expressed both separately and coordinately: characterization of the nrdB promoter

“…This time is taken as a working value for the in vivo appearance of active ribonucleoside diphosphate reductase (13,45). This enzyme has been detected in extracts at about 5 min after infection (53), but its kinetics of formation is much different and much faster than the initial exponential rise in the rate of synthesis of the deoxyribonucleotides (13,45,51); the kinetics of the latter is presumed to be dependent on the rate of formation of the dNTP synthetase complex (11,34,41,52), which contains ribonucleotide reductase as a central component (the term dNTP synthetase complex is an amalgamation of earlier designations used in our publications and in those of C. K. Mathews and co-workers [1,11,34,45,51]). …”

Are DNA precursors concentrated at replication sites?

“…We believe that such gradients are generated, at least in T4-infected bacteria, by the action of a multienzyme complex that synthesizes dNTPs from more distal DNA precursors (6)(7)(8)(9)(10). This complex, as studied in vitro, behaves as though capable of maintaining as much as a 50-fold gradient of deoxyribonucleotide intermediates (7). The in vivo gradient which we have estimated from this study is considerably lower but, as stated earlier, this represents a minimal estimate.…”

“…(iii) The compartmentation may be brought about by the action of specific dNTP-synthesizing multienzyme complexes, which behave as though juxtaposed with the replication apparatus (6)(7)(8)(9)(10)(11); such complexes seem able, in vitro, to maintain concentration gradients of deoxyribonucleotide intermediates (7). (iv) DNA precursor concentrations represent a determinant ofthe fidelity ofDNA replication.…”