Search

Previous work from this laboratory has shown that the cytosine-containing T4 deoxyribonucleic acid (DNA) made by deoxycytidine triphosphatase (dCTPase) amber mutants is extensively degraded, and that nucleases controlled by genes 46 and 47 participate in this process. In this paper, we examine other consequences of a defective dCTPase. Included are studies of DNA synthesis and phage production, and of the control of both early and late protein synthesis after infection of Escherichia coli B with various T4 mutants defective in genes 56 (dCTPase), 42 (dCMP hydroxymethylase), 1 (deoxynucleotide kinase), 43 (DNA polymerase), 30 (polynucleotide ligase), 46 and 47 (DNA breakdown) or e (lysozyme). By varying the temperature of infection with a temperature-sensitive dCTPase mutant, we have been able to control intracellular dCTPase activity, and thus vary the cytosine content of the phage DNA. We have produced and characterized viable T4 phage in which cytosine replaces 20% of the 5-hydroxymethylcytosine (HMC) in the DNA. We present evidence which suggests that intact, cytosine-containing T4 DNA is much less efficient than is normal T4 DNA in directing the synthesis of tail-fiber antigen. Lysozyme production is much less affected by progressively decreasing dCTPase activity; however, complete substitution of cytosine is correlated with a depression of lysozyme synthesis greater than expected from the defective synthesis of DNA. Low but significant lysozyme synthesis is observed late after infection of E. coli B with T4 amber mutants defective in a number of genes controlling DNA synthesis. The “20% cytosine” T4 phage, once produced, can initiate an apparently normal infection at permissive temperatures; the synthesis of early enzymes, DNA, and phage does not appear to be impaired. Two roles for HMC in T4 DNA have been indicated previously: (i) involvement in host-controlled restriction of the phage, in which glucosylation of the hydroxymethyl group plays a crucial role (16, 29, 53, 58), and (ii) protection of vegetative DNA against phage-controlled nucleases, a protection not dependent on glucosylation (41, 66, 67). A third role is suggested by our present results: transcription of at least some late genes can occur only from HMC-containing DNA and not from cytosine-containing DNA.

The time-course of degradation of bacterial DNA after infection of Escherichia coli B with bacteriophage T4 has been followed by means of neutral and alkaline sucrose density-gradient centrifugation. This degradation is found to occur in two stages. The first stage involves limited endonucleolytic cleavage of the DNA molecule, causing a gradual decrease in average molecular weight to a minimum of about 10 6 . There are a few single-strand breaks per double-strand fragment early in this stage but not later, indicating that both strands are broken at or near the same time. This first stage of the degradation still proceeds normally when genes 46 and/or 47 are defective. A second nuclease system is necessary to degrade these large DNA fragments to acid-soluble form. This second stage of the degradation proceeds rapidly once a fragment is attacked; little or no acid-insoluble material below a molecular weight of about 10 6 is found. Mutations in either gene 46 or 47 completely block this stage, leaving all of the bacterial DNA as fragments with a molecular weight of about 10 6 . The implications of these findings are discussed with respect to known nucleases, the phage-directed inhibition of host nucleic acid synthesis, and genetic recombination. Evidence is presented that the degradation of newly synthesized T4 DNA which occurs in infected cells lacking the phage-directed dCTPase occurs in two similar stages; in such cells cytosine is substituted extensively for the hydroxymethylcytosine normally found in T4 phage DNA. We have not determined whether the first stage of the degradation utilizes the same enzymes or factors on both host and cytosine-containing phage DNA, nor whether these enzymes are primarily of bacterial origin or represent new, phage-direeted functions. However, our evidence does establish that T4 genes 46 and 47 are essential to the second stage of degradation of both types of DNA. Although the cytosine-containing DNA produced by a triple mutant defective in genes 56 (dCTPase), 46 and 47 is not rendered acid-soluble by the cell, it is fragmented to a molecular weight of about 10 7 ; this is a sufficient explanation, though not the only one, for the failure of this mutant to produce phage particles. Possible explanations for the apparent cytosine specificity in the degradation of DNA are discussed; an attractive possibility is that the first enzyme to act on both host and phage DNA may be an endonuclease that attacks only at cytosinerich clusters.

Hydroxyurea (HU) has been used successfully (Warner el al. , 1970; Hercules et al. , 1971) to select bacteriophage T4 mutants which are deficient in degrading Escherichia coli DNA after infection. Using a different set of plating conditions, HU has also been used to isolate another set of T4 mutants ( hus ) ( Goscin and Hall, 1972 ). All the backcrossed hus mutants degrade bacterial DNA normally. The original hus 13 contained another mutation N. The double mutant hus 13-N degrades E. coli DNA slowly in the absence of phage DNA synthesis, leaving DNA fragments with a molecular weight midway between the sizes of fragments left by mutants defective in genes 46 and 47 and by endonuclease II ( den A) mutants at similar times after infection. Endonuclease IV activity is normal in hus 13-N infected cells. The HU sensitivity of three mutants, hus 3 (which is deficient in exonuclease A), hus 7 and hus 23 is partially due to inefficient utilization of host-derived deoxyribonucleotides as substrates for phage DNA synthesis. This is related to the DNA delay phenotype of these 3 mutants. The mutants hus 7 and hus 23 also appear to package phage DNA into phage particles inefficiently. The backcrossed hus 13 is apparently HU sensitive due to a defect in packaging DNA into phage particles. The HU sensitivity of two other mutants ( hus 19 and hus 20) remains unexplained as these mutants degrade bacterial DNA normally, synthesize phage DNA normally using host-derived deoxyribonucleotides, and package the DNA into phage particles efficiently.