Specific Suppression of Mutations in Genes 46 and 47 by das, a New Class of Mutations in Bacteriophage T4D

Mutants in T4 genes 46 and 47 exhibit early cessation of deoxyribonucleic acid (DNA) synthesis ("DNA arrest") and decreased synthesis of late proteins and phage. In addition, mutants in genes 46 and 47 fail to degrade host DNA to acidsoluble products. It is shown here that this complex phenotype can be partially suppressed by mutation of a T4 gene external to genes 46 and 47 which has been named das for "DNA arrest suppressor." The das mutations were discovered as third-site mutations in spontaneous pseudorevertants of [46, 47] mutants; the pseudorevertants make small plaques on Escherichia coli B, whereas [46, 47] mutants make none. The [das, 46, 47] triple mutant exhibits increased DNA, late protein, and viable phage production compared to the double mutant [46, 47]. The [das, 46, 47] mutant also degrades more of the host DNA to acid-soluble products than does the [46, 47] mutant. The suppressor effect of the das mutation appears to be gene-specific: it suppresses both amber and temperature-sensitive mutations in genes 46 and 47 and does not suppress amber mutations in any of the other genes tested. The [das] single mutants make normal-sized plaques on E. coli B and exhibit nearly normal host DNA degradation, DNA synthesis, late protein synthesis, and viable phage production. The das mutations either define a new gene between genes 33 and 34 or are special mutations within gene 33.     

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.     

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.     

Replication and Recombination of Gene 59 Mutant of Bacteriophage T4D

After infection of Escherichia coli B with phage T4D carrying an amber mutation in gene 59, recombination between two rII markers is reduced two- to three-fold. This level of recombination deficiency persists even when burst size similar to wild type is induced by the suppression of the mutant DNA-arrest phenotype. In the background of two other DNA-arrest mutants in genes 46 and 47, a 10- to 11-fold reduction in recombination is observed. The cumulative effect of gene 59 mutation on gene 46-47 mutant suggests that complicated interactions must occur in the production of genetic recombinants. The DNA-arrest phenotype of gene 59 mutant can be suppressed by inhibiting the synthesis of late phage proteins. Under these conditions, DNA replicative intermediates similar to those associated with wild-type infection are induced. Synthesis of late phage proteins, however, results in the degradation of mutant 200S replicative intermediate into 63S DNA molecules even in the absence of capsid assembly. Although these 63S molecules are associated with membrane, they do not replicate. These results suggest a role for gene 59 product, in addition to a possible requirement of concatemeric DNA in late replication of phage T4 DNA.     

Double-strand break repair in bacteriophage T4: coordination of DNA ends and effects of mutations in recombinational genes

Coordination of DNA ends during double-strand break (DSB) repair was studied in crosses of bacteriophage T4 in which DSBs were induced site-specifically by SegC endonuclease in the DNA of only one of the parents. Coupling of the genetic exchanges to the left and to the right of the DSB was measured in the wild-type genetic background as well as in T4 strains bearing mutations in several recombination genes: 47, uvsX, uvsW, 59, 39 and 61. The observed quantitative correlation between the degree of coupling and position of the recombining markers in relation to the DSB point implies that the two variants of the splice/patch-coupling (SPC) pathway, the "sequential SPC" and the "SPC with fork collision", operate during DSB repair. In the 47 mutant with or without a das suppressor, coupling of the exchanges was greatly reduced, indicating a crucial role of the 47/46 complex in coupling of the genetic exchanges on the two sides of the DSB. From the observed dependence of the apparent coupling on the intracellular ratio of breakable and unbreakable chromosomes in different genetic backgrounds it is inferred that linking of the DNA ends by 47/46 protein is the mechanism that accounts for their concerted action during DSB repair. A mechanism of replicative resolution of D-loop intermediate (RR pathway) is suggested to explain the phenomenology of DSB repair in DNA arrest and uvsW mutants. A "left"-"right" bias in the recombinogenic action of two DNA ends of the broken chromosome was observed which was particularly prominent in the 59 (41-helicase loader) and 39 (topoisomerase) mutants. Phage topoisomerase II (gp39-52-60) is indispensable for growth in the DNA arrest mutants: the doubles 47(-)39(-), uvsX 39(-) and 59(-)39(-) are lethal.     

Bacteriophage T4 Inhibits Colicin E2-Induced Degradation of Escherichia coli Deoxyribonucleic Acid I. Protein Synthesis-Dependent Inhibition

The deoxyribonucleic acid (DNA) of Escherichia coli B is converted by colicin E2 to products soluble in cold trichloroacetic acid; we show that this DNA degradation (hereafter termed solubilization) is subject to inhibition by infection with bacteriophage T4. At least two modes of inhibition may be differentiated on the basis of their sensitivity to chloramphenicol. The following observations on the inhibition of E2 by phage T4 in the absence of chloramphenicol are described: (i) Simultaneous addition to E. coli B of E2 and a phage mutated in genes 42, 46, and 47 results in a virtually complete block of the DNA solubilization normally induced by E2; the mutation in gene 42 prevents phage DNA synthesis, and the mutations in genes 46 and 47 block a late stage of phage-induced solubilization of host DNA. (ii) This triple mutant inhibits equally well when added at any time during the E2-induced solubilization. (iii) Simultaneous addition to E. coli B of E2 and a phage mutated only in gene 42 results in extensive DNA solubilization, but the amount of residual acid-insoluble DNA (20 to 25%) is more characteristic of phage infection than of E2 addition (5% or less). (iv) denA mutants of phage T4 are blocked in an early stage (endonuclease II) of degradation of host DNA; when E2 and a phage mutated in both genes 42 and denA are added to E. coli B, extensive solubilization of DNA occurs with a pattern identical to that observed upon simultaneous addition of E2 and the gene 42 mutant. (v) However, delaying E2 addition for 10 min after infection by this double mutant allows the phage to develop considerable inhibition of E2. (vi) Adsorption of E2 to E. coli B is not impaired by infection with phage mutated in genes 42, 46, and 47. In the presence of chloramphenicol, the inhibition of E2 by the triple-mutant (genes 42, 46, and 47) still occurs, but to a lesser extent.     

Late gene function in bacteriophage T4 in the absence of phage DNA replication

Under certain conditions the late genes of coliphage T4 may function in the absence of phage DNA replication. Quasi-late gene function is the function of certain late genes in the absence of both phage DNA replication and the product of the maturation gene 55. It does not depend on how phage DNA synthesis is prevented. Replication-uncoupled late gene function is late gene function from unreplicated DNA in the absence of phage ligase, and is still under the control of gene 55. It is most efficient if phage DNA replication is prevented by a mutation in the phage gene (43) for DNA polymerase. Both quasi-late gene function and replication-uncoupled late gene function are enhanced by the presence of mutations controlling a phage exonuclease (gene 46 or 47).     

Gene 32 protein of bacteriophage T4 moderates the activities of the T4 gene 46/47-controlled nuclease and of the Escherichia coli RecBC nuclease in vivo.

Genes 46 and 47 of phage T4 control a nuclease that is required for genetic recombination and may act similarly to the Escherichia coli RecBC nuclease. In vivo, the nucleolytic activities of both of these nucleases must be moderated so that recombining DNA intermediates are not destroyed. We conclude from our present experiments that the phage T4 gene 32 protein, specifically its C-terminal domain, participates in such moderation. We have investigated DNA degradation in different gene 32 and gene 32/46 mutants under conditions that are completely restrictive for progeny production in all the mutants. Under these conditions, DNA of those gene 32 mutants in which the C-terminal domain of the protein is not synthesized or is modified is degraded to acid-soluble material. T4 gene 46 or E. coli recB mutations reduce such degradation; together they abolish it completely. By contrast, single gene 32 mutants which produce an unaltered C-terminal domain show little or no degradation of their DNA. Residual protection against nucleases is unrelated to residual primary DNA replication or to overproduction of the mutant peptides in the different gene 32 mutants.     

Bacteriophage T4 Endonucleases II and Iv, Oppositely Affected by Dcmp Hydroxymethylase Activity, Have Different Roles in the Degradation and in the RNA Polymerase-Dependent Replication of T4 Cytosine-Containing DNA

Bacteriophage T4 mutants defective in gene 56 (dCTPase) synthesize DNA where cytosine (Cyt) partially or completely replaces hydroxymethylcytosine (HmCyt). This Cyt-DNA is degraded in vivo by T4 endonucleases II and IV, and by the exonuclease coded or controlled by genes 46 and 47.-Our results demonstrate that T4 endonuclease II is the principal enzyme initiating degradation of T4 Cyt-DNA. The activity of endonuclease IV, but not that of endonuclease II, was stimulated in the presence of a wild-type dCMP hydroxymethylase, also when no HmCyt was incorporated into phage DNA, suggesting the possibility of direct endonuclease IV-dCMP hydroxymethylase interactions. Endonuclease II activity, on the other hand, was almost completely inhibited in the presence of very small amounts of HmCyt (3-9% of total Cyt + HmCyt) in the DNA. Possible mechanisms for this inhibition are discussed.-The E. coli RNA polymerase modified by the products of T4 genes 33 and 55 was capable of initiating DNA synthesis on a Cyt-DNA template, although it probably cannot do so on an HmCyt template. In the presence of an active endonuclease IV, Cyt-DNA synthesis was arrested 10-30 min after infection, probably due to damage to the template. Cyt-DNA synthesis dependent on the unmodified (33-55-) RNA polymerase was less sensitive to endonuclease IV action.     

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.     

The pathway of recombination in phage T4

Summary A study has been made of the effect of modifying the products of the early T4 genes on the frequency with which haploid segregants are generated by recombination from a phage harbouring a standard genetic duplication. Alterations in the products of genes 32, 44, 46, 47 and 59 have been found to significantly decrease the segregation frequency and are, therefore, considered to be involved in the T4 recombination pathway.     

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 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.     

Recombination-dependent replication of plasmids during bacteriophage T4 infection

The replication of plasmids containing fragments of the T4 genome, but no phage replication origins, was analyzed as a possible model for phage secondary (recombination-dependent) replication initiation. The replication of such plasmids after T4 infection was reduced or eliminated by mutations in several phage genes (uvsY, uvsX, 46, 59, 39, and 52) that have previously been shown to be involved in secondary initiation. A series of plasmids that collectively contain about 60 kilobase pairs of the T4 genome were tested for replication after T4 infection. With the exception of those known to contain tertiary origins, every plasmid replicated in a uvsY-dependent fashion. Thus, there is no apparent requirement for an extensive nucleotide sequence in the uvsY-dependent plasmid replication. However, homology with the phage genome is required since the plasmid vector alone did not replicate after phage infection. The products of plasmid replication included long concatemeric molecules with as many as 35 tandem copies of plasmid sequence. The production of concatemers indicates that plasmid replication is an active process and not simply the result of passive replication after the integration of plasmids into the phage genome. We conclude that plasmids with homology to the T4 genome utilize the secondary initiation mechanism of the phage. This simple model system should be useful in elucidating the molecular mechanism of recombination-dependent DNA synthesis in phage T4.     

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.     

Role of recombinational repair in sensitivity to an antitumour agent that inhibits bacteriophage T4 type II DNA topoisomerase

The bacteriophage T4-encoded type II DNA topoisomerase is the major target for the antitumour agent m-AMSA (4-(9-acridinylamino)methanesulphon-m-anisidide) in phage-infected bacterial cells. Inhibition of the purified enzyme by m-AMSA results in formation of a cleavage complex that contains the enzyme covalently attached to DNA on both sides of a double-strand break. In this article, we provide evidence that this cleavage complex is responsible for inhibition of phage growth and that recombinational repair can reduce sensitivity to the antitumour agent, presumably by eliminating the complex (or some derivative thereof). First, topoisomerase-deficient mutants were shown to be resistant to m-AMSA, indicating that m-AMSA inhibits growth by inducing the cleavage complex rather than by inhibiting enzyme activity. Second, mutations in several phage genes that encode recombination proteins (uvsX, uvsY, 46 and 59) increased the sensitivity of phage T4 to m-AMSA, strongly suggesting that recombination participates in the repair of topoisomerase-mediated damage. Third, m-AMSA stimulated recombination in phage-infected bacterial cells, as would be expected from the recombinational repair of DNA damage. Finally, m-AMSA induced the production of cleavage complexes involving the T4 topoisomerase within phage-infected cells.     

Recombination in amber-mutants of bacteriophage T4B. I. Recombination and complementation in phage T4 amber-mutants]

In studying intergenic and intragenic complementation in amber mutants in genes of phage T4 controlling the synthesis of phage tail fibres the data have been obtained indicating the dependency of the results of complementation tests on those of crosses of respective markers. The results obtained show that in complementation of amber mutants of phage T4 the phage yield varies widely and depends on the location of markers on the phage genetic map.     

Fate of cloned bacteriophage T4 DNA after phage T4 infection of clone-bearing cells

Plasmid pBR322 replication is inhibited after bacteriophage T4 infection. If no T4 DNA had been cloned into this plasmid vector, the kinetics of inhibition are similar to those observed for the inhibition of Escherichia coli chromosomal DNA. However, if T4 DNA has been cloned into pBR322, plasmid DNA synthesis is initially inhibited but then resumes approximately at the time that phage DNA replication begins. The T4 insert-dependent synthesis of pBR322 DNA is not observed if the infecting phage are deleted for the T4 DNA cloned in the plasmid. Thus, this T4 homology-dependent synthesis of plasmid DNA probably reflects recombination between plasmids and infecting phage genomes. However, this recombination-dependent synthesis of pBR322 DNA does not require the T4 gene 46 product, which is essential for T4 generalized recombination. The effect of T4 infection on the degradation of plasmid DNA is also examined. Plasmid DNA degradation, like E. coli chromosomal DNA degradation, occurs in wild-type and denB mutant infections. However, neither plasmid or chromosomal degradation can be detected in denA mutant infections by the method of DNA--DNA hybridization on nitrocellulose filters.     

Persistence of DNA synthesis arrest sites in the presence of T4 DNA polymerase and T4 gene 32, 44, 45 and 62 DNA polymerase accessory proteins.

DNA synthesis by phage T4 DNA polymerase is arrested at specific sequences in single-stranded DNA templates. To determine whether or not T4 DNA polymerase accessory proteins 32, 44, 45 and 62 eliminated recognition of these arrest sites, unique primer-templates were constructed in which DNA synthesis began at a DNA primer located at different distances from palindromic and nonpalindromic arrest sites. Nucleotide positions that caused polymerase to pause or leave the template were identified by sequence analysis of 5'-end labeled nascent DNA chains. Stable hairpin structures at palindromic sequences were confirmed by acetylation of single-stranded sequences with bromoacetaldehyde. Our results confirmed that these T4 DNA polymerase accessory proteins stimulated T4 DNA polymerase activity and processivity on natural as well as homopolymer primer-templates. However, they did not alter recognition of DNA synthesis arrest sites by T4 DNA polymerase. Extensive DNA synthesis resulted from an increased rate of translocation and/or processivity to the same extent over all DNA sequences.