In order to retain in an in situ system the control mechanisms involved in synthesis of bacteriophage T4 DNA, infected cells were made permeable to nucleotides by plasmolysis with concentrated sucrose. Such preparations use exogenous deoxyribonucleotides to synthesize T4 phage DNA. As has been observed with in vivo studies, DNA synthesis was drastically reduced in plasmolyzed preparations from cells infected by amber mutants of genes 1, 32, 41, 42, 43, 44, or 45. Added 5-hydroxymethyl dCTP did not bypass either a mutant of gene 42 (dCMP hydroxymethylase) or of gene 1 (phage-induced deoxyribonucleotide kinase). In a phage system lacking deoxycytidine triphosphatase (gene 56) and the gene-46 product, and therefore incorporating dCTP into DNA, dCTP incorporation did not require dCMP hydroxymethylase, in keeping with in vivo results. With a triple amber mutant of genes 1, 46, and 56 only slight incorporation of dCTP occurred. By contrast, in experiments performed in vivo the synthesis of cytosine-containing DNA was unaffected by an amber mutation in gene 1.These studies provide evidence that dCMP hydroxymethylase, in addition to its known catalytic function, has a second, more direct, role in phage T4 DNA synthesis, apparently in recognition of hydroxymethyl dCTP. The role of the phage-induced deoxyribonucleotide kinase in T4 DNA synthesis in the plasmolyzed system remains unresolved.While the work of several laboratories (1-5) has established the intimate role of phage T4-induced DNA polymerase in the synthesis of T4 DNA, it has long been clear that several other factors are required. For example, DNA ligase (gene 30) (6, 7), an unwinding protein (gene 32) (8), and other undefined components participate in the process (9, 10). Clearly, DNA synthesis in Escherichia coli requires several factors as well (11).As a result, the concept that a complex of enzymes or structural components is required for DNA synthesis both in phage T4 infection and in bacterial systems has been conconsidered (9, 11-13). Alberts and coworkers, using a concentrated extract prepared from T4-infected cells, have shown a requirement in DNA synthesis for the products of genes 41, 44, 45, and 62 (14). An interaction has been found between the products of genes 44 and 62 (15) and also between phage T4 DNA unwinding protein (gene 32) and T4 DNA polymerase (gene 43) (16).Though the apparent involvement of the undefined DO (no DNA synthesis) gene products has been widely recognized, it has been generally considered that DNA synthesis proceeds quite independently of the pathways leading to its deoxyribonucleotide precursors, except insofar as these pathways become limiting (Fig. 1). On the basis of an investigation of a temperature-sensitive mutant, Chiu and Greenberg suggested that the phage T4-induced enzyme, dCMP hydroxymethylase (gene 42), in addition to catalyzing the formation of 5-hydroxymethyl dCMP, also plays a direct role in DNA replication (13). We now have tested this possibility in phageinfected cells rendered permeable by plasmo...

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...

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Bacteriophage T4-infected Escherichia coli rendered permeable to nucleotides by sucrose plasmolysis exhibited two apparently separate pathways or channels to T4 DNA with respect to the utilization of exogenously supplied substrates. By one pathway, individual labeled ribonucleotides, thymidine (TdR), and 5-hydroxy-methyl-dCMP could be incorporated into phage DNA. Incorporation of each of these labeled compounds was not dependent upon the addition of the other deoxyribonucleotide precursors, suggesting that a functioning de novo pathway to deoxyribonucleotides was being monitored. The second pathway or reaction required all four deoxyribonucleoside triphosphates or the deoxyribonucleoside monophosphates together with ATP. However, in this reaction, dTTP was not replaced by TdR. The two pathways were also distinguished on the basis of their apparent Mg2+ requirements and responses to N-ethylmaleimide, micrococcal nuclease, and to hydroxyurea, which is a specific inhibitor of ribonucleoside diphosphate reductase. Separate products were synthesized by the two channels, as shown by density-gradient experiments and velocity sedimentation analysis. Each of the pathways required the products of the T4 DNA synthesis genes. Furthermore, DNA synthesis by each pathway appeared to be coupled to the functioning of several of the phage-induced enzymes involved in deoxyribonucleotide biosynthesis. Both systems represent replicative phage DNA synthesis as determined by CsCl density-gradient analysis. Autoradiographic and other studies provided evidence that both pathways occur in the same cell. Further studies were carried out on the direct role of dCMP hydroxymethylase in T4 DNA replication. Temperature-shift experiments in plasmolyzed cells using a temperature-sensitive mutant furnished strong evidence that this gene product is necessary in DNA replication and is not functioning by allowing preinitiation of DNA before plasmolysis.

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