Simultaneous initiation of synthesis of bacteriophage T4 DNA and of deoxyribonucleotides.

Abstract

In earlier reports we have suggested that bacteriophage T4 DNA replication occurs in a complex composed of the proteins required for polymerization and the system of enzymes synthesizing the deoxyribonucleoside triphosphate precursors of DNA. T4-induced dCMP hydroxymethylase and dTMP synthetase, though demonstrable in extracts soon after infection, are not active in vivo until about 5 min. The in vivo activities increase exponentially for approximately 15 min and then become constant. We have suggested that the exponential period represents the formation of the complexes.This paper shows that the initiation of DNA synthesis and of the two deoxyribonucleotide-synthesizing activities occurs simultaneously and with coinciding exponential kinetics.The in vivo activities of the two enzymes were tested after infection by a number of T4 amber Dna-mutants. Their activities were essentially unchanged compared to the wildtype phage, except on infection by mutants of gene 43 (T4 DNA nucleotidyltransferase or DNA polymerase). With these mutants the rate of increase of dTMP synthetase and dCMP hydroxymethylase activities was always substantially lower than after infection by wild-type phage. It is proposed that an intimate interaction occurs between T4-induced DNA polymerase and the complex of enzymes forming 5-hydroxymethyl-dCMP and dTMP. Infection by bacteriophage T4 induces a series of clocked events leading ultimately to the initiation of phage DNA synthesis. To this end a large body of work has described the control of early messenger RNAs (1-3) and the subsequent formation of the early enzymes (4). It has been generally accepted that DNA synthesis occurs only after formation of the enzymes required for its replication (5) and for the synthesis of its deoxyribonucleotide substrates. However, we have developed evidence that the regulatory process is more sophisticated. Thus certain early enzymes, namely, dCMP hydroxymethylase and 5-hydroxymethyl-dCMP (hmdCMP) kinase, have dual functions (6). That is, they not only form deoxyribonucleotide products, but also participate directly in the DNA replication process. Other laboratories have reported similar findings (7,8). More recently we have shown that at 300 dCMP hydroxymethylase and phage-induced dTMP synthetase, though formed 1-2 min and 3-4 min after infection, respectively, are not active in vivo until about 4.8 min (9). Beginning at this time the activities of these enzymes increase exponentially until about 20 min and then become constant. We have proposed that these enzymes and apparently others must form a complex in order to function in vivo and that the exponential kinetics represent the formation of this complex(es) from its limiting component parts. The number of these complexes ultimately determines the rate of synthesis of the deoxyribonucleotides and then of DNA. Implicit in our earlier study was the concept that the time of initiation of T4 DNA synthesis and the initial exponential kinetics coincide with the initiation and the kinetics of activation...

Role of the Dna/Membrane Complex in Prokaryotic Dna Replication

“…Membrane association might help supply energy to the initiation complex and channel immediate DNA precursors to the membrane-associated replication apparatus (88). It has long been known that a T4 deoxynucleotide synthetase is involved in supplying immediate DNA precursors to the replisome (12,79,97) and that one of the enzymes in this complex, dCTPase, is also encoded by the oriA region (88). Thus, the gp69 membrane protein can be added to the list of known mem brane-associated replication proteins.…”

Selective screening procedure for the isolation of heat- and cold-sensitive, DNA replication-deficient mutants of bacteriophage SPO1 and preliminary characterization of the mutants isolated

“…It does not seem likely that this could be explained simply by invoking a smaller hydroxymethyl dUMP pool size for hs29-2, since the HMase activity of extracts of hs29-2-infected cells was as great at both 30 and 430C as that of hs29-1, which shuts off replication slowly (Cregg, personal communication). Greenberg and co-workers (1,27) have argued that dCMP HMase, the equivalent enzyme in T4 infection, is part of a complex that functions both for nucleotide synthesis and directly in DNA polymerization. The existence of a similar complex in SPOl-infected B. subtilis could explain the varying phenotypes of cistron 29 mutants, with those in classes I and II affecting only the nucleotide synthesis function, whereas those in class Ill affect the polymerization function.…”

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

“…As determined by the very sensitive tritium release assay, deoxyribonucleotide synthesis initiates at 4.8 min after infection at 30°C. 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]).…”

“…In turn, the onset of phage DNA replication, which occurs at about 5 min after infection, is dependent on the turning on of deoxyribonucleotide synthesis (13,33,45,51). Accordingly, workers in our laboratory are investigating the control of expression of the T4 nrdA and nrdB genes, which encode the a2 and 2 subunits, respectively, of ribonucleotide reductase (4,5,16,36,39,43,46).…”