Studies on the Recombination Genes of Bacteriophage T4: Suppression of uvsX and uvsY Mutations by uvsW Mutations

Genes uvsW, uvsX and uvsY are dispensable for T4 growth but are implicated in recombination and in the repair of damaged DNA. We found that large-plaque mutants arose efficiently from small-plaque uvsX and uvsY mutants at 42 degrees and were pseudorevertants containing a new mutation in uvsW. Using reconstructed double mutants, we confirmed that a mutation in uvsW partially increases the burst size and UV resistance of uvsX and uvsY mutants. At 41 degrees the uvsW mutation completely restores the arrest in DNA synthesis caused by mutations in genes uvsX, uvsY and 46, but at 30 degrees it only partially restores DNA synthesis in a gene 46 mutant and does not restore DNA synthesis in uvsX and uvsY mutants. Restored DNA synthesis at 41 degrees was paralleled by the overproduction of single-stranded DNA and gene 32 protein. Based on these findings, we propose that the uvsW gene regulates the production of single-stranded DNA and we discuss the phenotype of uvsW mutants and their suppression of some uvsX and uvsY phenotypes. Infection of restrictive cells with am uvsW mutants revealed a defect in the synthesis of a protein of molecular weight 53,000 daltons, suggesting that this protein is the uvsW gene product.     

Effect of a Gene-Specific Suppressor Mutation (das) on DNA Synthesis of Gene 46-47 Mutants of Bacteriophage T4D

Mutants in genes 46 and 47 of bacteriophage T4 exhibit early cessation of DNA synthesis, inability to form a normal rapidly sedimenting DNA intermediate (200S), reduced genetic recombination, and reduced viable phage production. A gene-specific suppressor mutation called das partially restores many of the pleiotropic effects of gene 46-47 mutants (13). Our results indicate that this partial suppression by das is associated with (i) the synthesis of a small fraction of DNA containing long single chains not detectable in 46-47 infection and (ii) a decrease in an "early" function which participates in the degradation of DNA synthesized in the absence of 46-47 functions. However, das does not restore the formation of a normal rapidly sedimenting (200S) DNA intermediate.     

Suppression of Adsorption Properties of a z Mutant of Bacteriophage T4 by r Mutations

The products of bacteriophage T4 r genes influence the organization of the phage-adsorption apparatus in a noncatalytic way.     

Role of Genes 46 and 47 in Bacteriophage T4 Reproduction: I. In Vivo Deoxyribonucleic Acid Replication

Functional proteins coded by genes 46 and 47 are required for (i) continuation of deoxyribonucleic acid (DNA) synthesis in the late period of T4 infection and (ii) production of normal, late replicating DNA which contains strands with a sedimentation coefficient in alkaline sucrose greater than that of mature DNA (73S). Continued DNA synthesis in the late period in the absence of functional genes 46 or 47 can be achieved by inhibiting late protein synthesis either by using bacterio-phage with a second mutation in gene 55 or by adding chloramphenicol to the culture before the decline in the rate of DNA synthesis. However, when functional 46/47 proteins are absent throughout infection, no strands with a sedimentation coefficient greater than 73S (in alkaline sucrose) are produced. This is the case even when DNA synthesis is allowed to continue. DNA arrest is accompanied by conversion of rapidly sedimenting, replicating DNA to slower sedimenting forms. When 46/47 is absent from the beginning of infection, the conversion product has a smaller sedimentation coefficient than mature DNA both in neutral and alkaline sucrose. When DNA arrest occurs midway in infection by heat-inactivating the ts46 enzyme, the conversion product has a sedimentation coefficient (i) the same as mature DNA in both neutral (63S) and alkaline sucrose if capsid assembly is allowed to take place and (ii) close to 63S in neutral sucrose but heterogenous and relatively greater (up to 100S) in alkaline sucrose if capsid assembly is inhibited. The structure of this DNA is unknown.     

Reversion of Frameshift Mutations Stimulated by Lesions in Early Function Genes of Bacteriophage T4

Temperature-sensitive (ts) mutants representative of a number of genes of phage T4 were crossed with rII mutants to allow isolation of ts, rII double-mutant recombinants. The rII mutations used were characterized as frameshift mutations primarily on the basis of their revertability by proflavine. For each ts, rII double mutant, the effect of the ts mutation on spontaneous reversion of the rII mutation was determined over a range of incubation temperatures. A strong enhancement in reversion of two different rII mutants was detected when they were combined with tsL56, a mutation in gene 43 [deoxyribonucleic acid (DNA) polymerase]. Three other mutants defective in gene 43 enhanced reversion about fourfold. Two mutations in gene 32, which specifies a protein necessary for DNA replication, enhanced reversion about 5-fold and 18-fold, respectively. Two additional mutations in gene 43 and two in gene 32 had no effect. Fivefold and threefold enhancements in reversion were also found with mutations in genes 44 (DNA synthesis) and 47 (deoxyribonuclease), respectively. No significant effect was found with mutations in seven additional genes. The results of other workers suggest that frameshift mutations arise from errors in strand alignment during repair synthesis occurring at chromosome tips. Our results show that such errors can be enhanced by mutations in the DNA polymerase, the gene 32 protein, and the enzymes specified by genes 44 and 47. This implies that these proteins are employed in the repair process occurring at chromosome tips and that mutational errors in these proteins can lead to loss of ability to recognize and reject strand misalignments.     

Partial Suppression of Bacteriophage T4 Ligase Mutations by T4 Endonuclease II Deficiency: Role of Host Ligase

Endonuclease II-deficient, ligase-deficient double mutants of phage T4 induce considerably more deoxyribonucleic acid (DNA) synthesis after infection of Escherichia coli B than does the ligase-deficient single mutant. Furthermore, the double mutant can replicate 10 to 15% as well as wild-type T4, whereas the single mutant fails to replicate. When the E. coli host is also deficient in ligase, the double mutant resembles the single mutant. The results indicate that host ligase can substitute for phage ligase when the host DNA is not attacked by the phage-induced endonuclease II.     

Membrane-associated DNase activity controlled by genes 46 and 47 of bacteriophage T4D and elevated DNase activity associated with the T4 das mutation.

Lethal, amber mutations in T4 genes 46 and 47 cause incomplete degradation of host DNA, premature arrest of phage DNA synthesis, accumulation of abnormal DNA replication intermediates, and defective recombination. These phenotypes can be explained by the hypothesis that genes 46 and 47 control a DNA exonuclease, but in vitro demonstration of such a nuclease has not yet been reported. Membrane and supernatant fractions from 46- and 47- mutant-infected and 46+ 47+ control-infected cells were assayed for the presence of the protein products of these genes (i.e., gp46 and gp47) and for the ability to degrade various DNA substrates to acid-soluble products in vitro. The two proteins were found only on membranes. The membrane fraction from 46- 47- mutant-infected cells digested native or heavily nicked Escherichia coli DNA to acid-soluble products three to four times slower that the membrane fraction from control-infected cells. No such effect was found in the cytoplasmic fractions. The effect on nuclease activity in membranes was the same whether 46- and 47- mutations were present singly or together. NaClO4, a chaotropic agent, released both gp46 and gp47 from 46+ 47+ membranes, as well as the DNase activity controlled by genes 46 and 47. DNA cellulose chromatography of proteins released from membranes by NaClO4 showed that gp46 and gp47 bound to the native DNAs of both E. coli and T4. Thus, the overall enrichment of gp46 and gp47 relative to total T4 protein was 600-fold (10-fold in membranes, 2-fold more upon release from membranes by NaClO4, and 30-fold more upon elution from DNA cellulose). T4 das mutations, which partially suppress the defective phenotype of 46- and 47- mutants, caused a considerable increase in vitro DNase activity in both membrane and cytoplasmic fractions, We obtained evidence that the das+ gene does not function to inhibit E. coli exonuclease I or V, endonuclease I, or the UV endonuclease of gene uvrA or to decrease the activity of T4 exonuclease A or the T4 gene 43 exonuclease.     

Gene Expression and Stability of mRNA Affected by DNA-Arrested Synthesis in Gene 59, 46, and 47 Mutants of Bacteriophage T4

The effect of bacteriophage T4 gene 59 mutations (DNA-arrested synthesis) on kinetics of DNA synthesis, gene expression, and stability of mRNA has been studied. When Escherichia coli B was infected by a T4 gene 59 mutant, DNA synthesis proceeded to increase linearly after initiation, but started to decrease at 8 min and was completely arrested at 12 min at 37°C. At various incubation temperatures (20 to 42°C), the initial rates and times of arrest of DNA synthesis were different, but the total amount of DNA synthesized was constant. This result supports the hypothesis that function of gene 59 is required for the conversion of 63S DNA molecules to other replicative intermediates (39). The abnormality in protein synthesis caused by gene 59 mutation is manifested by (i) a delayed shutoff in the expression of early proteins (gene 43, 46, 39, 52, 63, 42-45, and some unidentified proteins), (ii) a reduced rate of late gene expression (gene 34, 37, 18, 20, 23, wac, 24, 22, 38, and 19), and (iii) an absence of cleavage of certain late proteins (23, 24, IPIII and 22 to 23^*, 24^*, IPIII^*, and small fragments). It appears that there was no effect on the expression of gene 33, 55, and 32 by a mutation in gene 59. Results obtained from an addition of rifampin at the prereplicative cycle after infection indicated that mRNA from genes 43, rIIA, 46, 39, 52, and 63 are more stable in T4amC5 (gene 59) than in wild-type-infected cells. mRNA remained functional longer in mutant-infected cells, and this may explain the prolonged synthesis of certain early proteins. The gene expression of other DNA arrested mutants—those in genes 46 and 47—showed a pattern of abnormal protein synthesis similar to that found in gene 59 mutant-infected cells, except more late proteins are synthesized. The gene expression in terms of phage DNA structure is discussed.     

Variations in Genetic Recombination Due to Amber Mutations in T4D Bacteriophage

Recombination experiments were performed to assess the affect of amber mutations in 12 genes of T4D bacteriophage on genetic recombination. Crosses were performed in various suppressor-containing bacterial hosts to permit the production of progeny phage. Amber mutations in genes 32, 46, and 47 caused decreased recombination, amber mutations in genes 30, 41, 42, 43, 56, 61, and 62 caused increased recombination, whereas mutations in genes 63 and 37 showed no demonstrable effect on recombination.     

Suppression of Amber Mutations of Bacteriophage T4 Gene 43 (DNA Polymerase) by Translational Ambiguity

The growth properties of twelve different amber (am) mutants of bacteriophage T4 gene 43 (DNA polymerase) were examined by using nonpermissive (su(-)) as well as permissive (su(+)) Escherichia coli hosts. It was found that most of these mutants were measurably suppressed in su(-) hosts by translational ambiguity (misreading of codons during protein synthesis). The ability of these mutants to grow in response to this form of weak suppression probably means that the T4 gene 43 DNA polymerase can be effective in supporting productive DNA replication when it is supplied in small amounts. By similar criteria, studies with other phage mutants suggested that the products of T4 genes 62 (uncharacterized), 44 (uncharacterized), 42 (dCMP-hydroxymethylase), and 56 (dCTPase) are also effective in small amounts. Some T4 gene products, such as the product of gene 41 (uncharacterized), seem to be partially dispensable for phage growth since am mutants of such genes do propagate, although weakly, in streptomycin-resistant su(-) hosts which appear to have lost the capacity to suppress am mutations by ambiguity.     

Some Acridine-Resistant Mutations of Bacteriophage T4d

Three new 9-aminoacridine (9AA) resistant mutations of bacteriophage T4D have been isolated and characterized. Two of the mutations, rs and rc, have identical patterns of acridine resistance, but they map on opposite sides of the rII region. In addition, rs has an effect on the plaque morphology of r mutations, whereas rc does not. The third mutation, ama, maps very close to rs but exhibits a different pattern of resistance to 9AA. None of the three is resistant to acridines by virtue of reduced permeability. Taken together with other mutations that have been previously characterized, these new mutations permit us to set the minimum number of acridine-sensitive processes in T4 development at four.     

Intragenic Complementation by Gene 42 Amber Mutations of Bacteriophage T4

Phage T4 amber mutants defective in gene 42 (dCMP hydroxymethylase) were shown by in vivo and in vitro experiments to participate in both positive and negative intragenic complementation. This argues that incomplete polypeptide chains can participate in subunit interaction.     

Suppression of DNA Arrest Mutants in Bacteriophage T4

A mutation in gene 49 of phage T4 was not able to restore DNA synthesis in a gene 46 mutant.     

Inactivation of T4D Bacteriophage by Antiserum Against Bacteriophage Dihydrofolate Reductase

Antiserum was prepared against highly purified T4D bacteriophage-induced dihydrofolate reductase (DFR). This serum not only inactivated the enzyme but also inactivated all strains of T4D examined. T6 was inactivated to a lesser extent, and T2L, T2H, and T5 were unaffected by the antiserum. The phage-killing power of the serum could be blocked by prior incubation with partially purified T4D dfr obtained from host cells unable to make phage structural proteins. These observations confirm earlier results that the phage dfr is a structural component of the phage particle, and they offer new evidence on the manner in which this enzyme in incorporated into the tail structure.     

Genes 55, alpha gt, 47 and 46 of bacteriophage T4: the genomic organization as deduced by sequence analysis

The nucleotide sequence of T4 genes 55, alpha gt, 47 and 46 was determined by a combination of 'classical' procedures and a shotgun approach. Small DNA fragments generated by frequent cleavage with restriction enzymes or by sonication of restriction fragments were cloned in phage M13 vectors and sequenced by the dideoxy method. The positions of the genes were determined by marker rescue between the corresponding T4 amber mutants and the cloned T4 DNA fragments used in the sequencing experiments. The sequence gives an insight into the organization of this 7.1-kb early region of the T4 genome and shows that genetically 'silent' portions within this region are not void of genetic information.     

Role of Gene 46 in Bacteriophage T4 Deoxyribonucleic Acid Synthesis

In an attempt to establish whether Escherichia coli B infected with N130 (an amber mutant defective in gene 46) is recombination-deficient, the postinfection fate of (14)C-labeled N130 parental deoxyribonucleic acid (DNA) was followed, its amount in complex with the host cell membrane being determined in sucrose gradients after mild lysis of the infected cells. The parental DNA was found to undergo gradual detachment from the membrane during infection. Pulse-chase experiments similarly showed that newly synthesized DNA is normally attached to the host cell membrane and is detached by endonucleolytic breakage at a late stage of infection. The conclusion is that only attached DNA molecules are replicated by membrane-bound replicase, whereas those detached by endonucleolytic breakage are not. It thus seems that the gene 46 product controls the activity of a nuclease whose main function is recombination of DNA nicked by endonuclease, thereby attaching it to the host cell membrane. The rate of T4 DNA synthesis is apparently governed by the efficiency of recombination. Supporting evidence was found in experiments with the double mutant N130 x N134 (genes 46, 33).