Packaging of coliphage lambda DNA. I. The role of the cohesive end site and the gene A protein

The cohesive ends of the DNA of bacteriophage λ particles are normally formed by the action of a nuclease on the cohesive end sites (cos) of concatemeric λ DNA (reviewed by Hohn et al., 1977). The nuclease also cuts the cos site of an integrated prophage, and DNA located to the right is preferentially packaged into phage particles. This process occurs with approximately the same efficiency and rate in a single lysogen as in a tandem polylysogen. Thus, the rate of cos cutting does not increase when the number of cos sites per molecule increases, an hypothesis that has been proposed to explain why cohesive ends are not formed in circular monomers of λ DNA. We propose instead that the interaction of Ter with cos is influenced by the configuration of the DNA outside of cos during packaging, and that this configuration is different for circular monomers than for other forms of λ DNA. A model that gives rise to such a difference is described.We also found that missense mutations in the λ A gene changed the efficiency of packaging of phage relative to host DNA. This was not the case for missense mutations in several phage genes required for capsid formation. Thus, the product of gene A plays a role in determining packaging specificity, as expected if it is or is part of the nuclease that cuts λ DNA at cos.     

Initiation of recA+-dependent recombination in Escherichia coli (lambda). I. Undamaged covalent circular lambda DNA molecules in uvrA+ recA+ lysogenic host cells are cut following superinfection with psoralen-damaged lambda phages.

When λ bacteriophages were treated with a photosensitizing agent, psoralen or khellin, and 360 nm light, monoadducts and interstrand crosslinks were produced in the phage DNA. The DNA from the treated phages was injected normally into Escherichia coli uvrA^? (λ) cells and it was converted to the covalent circular form in yields similar to those obtained in experiments with undamaged λ phages. In excision-proficient host cells, however, there was a dose-dependent reduction in the yield of rapidly sedimenting molecules, and a corresponding increase in slow sedimenting material, the extent of this conversion corresponding to about one cut per two crosslinks. Presumably, the damaged λ DNA molecules were cut by the uvrA endonuclease of the host cell, but were not restored to the original covalent circular form.The presence of psoralen damage in λ phage DNA greatly increased the frequency of genetic exchanges in λ phage-prophage crosses in homoimmune lysogens (Lin et al., 1977). As genetic recombination is thought to depend on cutting and joining in DNA molecules, experiments were performed to test whether psoralen-damaged λ DNA would cause other λ DNA in the same cell to be cut. E. coli (λ) host cells were infected with ³²P-labeled λ phages and incubated to permit the labeled DNA to form covalent circles. When these host cells were superinfected with untreated λ phages, there was no effect upon the circular DNA. When superinfected with λ phages that had been treated with psoralen and light, however, many of the covalent circular molecules were cut. The cutting of undamaged molecules in response to the damaged DNA was referred to as “cutting in trans”. It required the uvrA⁺ and recA⁺ host gene functions, but neither recB⁺ nor any phage gene functions. It occurred normally in non-lysogenic hosts treated with chloramphenicol before infection. Cutting in trans may be one of the steps in recA-controlled recombination between psoralen crosslinked phage λ DNA and its homologs.     

Bacteriophage P2-lambda interference. III. Essential role of an early step in the initiation of lambda replication.

Induction of bacteriophage λ in the presence of a P2 prophage results in inactivation of cellular transfer RNA, inhibition of amino acid and uridine incorporation in the host, as well as inhibition of phage replication. A red gam double mutation allows λ to escape from interference, and a mutation in gene O or P abolishes the effects on the host.It is shown here that phage and plasmid DNA extracted from cells undergoing P2-λ interference are still active in a transfection assay. Mutations in bacterial gene dna B or in phage site ori suppress the inhibition of amino acid incorporation, whereas genes dnaE and dna G have no such effect. Derepression of bacterial exonuclease VIII totally suppresses the interference, and mutations in genes recA and lexA, which control the SOS functions, suppress it partially if the λ phage is red⁺. Our results suggest that P2-λ interference is due to the action of old at an early step of the initiation of λ replication.     

The isolation and characterization of plaque-forming arabinose transducing bacteriophage lambda.

Non-defective arabinose transducing phage, λpara, were isolated in two steps: first, Escherichia coli strains containing rare insertions of λ DNA into the arabinose C or B genes were selected; and second, these lysogens were induced and transducing phage were selected from the resulting lysates. The approximate location of the bacterial substitution on the phage and the ara gene content of the substitution were determined genetically. The precise location of the substitution was determined by electron microscopy of DNA heteroduplexes.Transducing phage, derived from the strain possessing λ inserted into the araC gene, carried part of the araC gene, the ara regulatory region, and all of the araB gene. Transducing phage, derived from eight independent strains possessing λ inserted in the same orientation and in the same position in the araB gene, carried a portion of the araB gene, the ara regulatory region and all of the araC gene. In these nine cases the ara DNA on the phage was immediately adjacent to the normal phage attachment site, indicating that the transducing phage were formed by the same type of abnormal excision which produces gal or bio transducing λ phage. The relative orientations of ara and phage genes were deduced from the topology of such excisions. One anomalous transducing phage was also characterized.     

DNA packaging steps in bacteriophage lambda head assembly

Petit λ is an empty spherical shell of protein which appears wherever λ grows. If phage DNA and petit λ are added to a cell-free extract of induced lysogenic bacteria, then phage particles are formed that contain the DNA and protein from the petit λ. Petit λ is transformed, without dissociation, into a phage head by addition of DNA and more phage proteins.The products of ten genes, nine phage and one host, are required for λ head assembly. Among these, the products of four phage genes, E, B, C, and Nu3 and of the host gene groE are involved in the synthesis of petit λ, consequently these proteins are dispensable for head assembly in extracts to which petit λ has been added. The products of genes A and D allow DNA to combine with petit λ to form a head that has normal morphology. In an extract, DNA can react with A product and petit λ to become partially DNAase-resistant, as if an unstable DNA-filled intermediate were formed. ATP and spermidine are needed at this stage. This intermediate is subsequently stabilized by addition of D product. The data suggest a pathway for head assembly.     

On the lack of host-cell reactivation of UV-irradiated phage T5 II. Further characterization of the repair inhibition exerted by T5 infection

Experiments reported in the preceding paper [4] had shown that host-cell reactivation (HCR) of UV-irradiated phage T1 in excision-repair proficient Escherichia coli cells is inhibited by superinfection with phage T5. Theoretical considerations have led to predictions concerning the dependence of repair inhibition on the multiplicity of superinfecting T5 phage and on the UV fluence to which they were exposed. These predictions have been supported by experimental results described in this paper. The fluence dependence permitted calculation of the relative UV sensitivity of the gene function responsible for repair inhibition; it was found to be about 2.3% that of the plaque-forming ability of phage T5.The T5-inhibitable step in excision repair occurs early in the infective cycle of T1. Furthermore, experiments involving the presence of 400 μg/ml chloramphenicol showed that HCR inhibition of T1 is caused by a protein produced after the FST segment of T5 (i.e. the first 8% of the T5 genome) has entered the host cell. A previously described minor T1 recovery process, occuring in both excision-repair-proficient and -deficient host cells, is inhibited by T5 infection due to a different substance, which is most likely associated with the “second-step-transfer” region of T5 DNA (involving the remainder of the genome). Superinfection with T4ν₁ phage resulted in HCR inhibition of T1, resembling that observed after T5 superinfection. The discussion of these results suggests that inhibition of the bacterial excision repair system by T5 or T4 infection occurs at the level of UV-endonucleolytic incision, and that lack of HCR both in T-even phages and in T5 can be explained in the same manner.     

Initiation of synthesis of the structural proteins of Semliki Forest virus.

Insertion of phage λ DNA into the normal attachment site of the DNA of the host Escherichia coli has been studied by ultracentrifugation analysis of the conversion of covalent circles of F′450 (F′gal att^λ bio) to F′450(λ) circles. We have found that integration proceeds at the normal rate if, in addition to the int gene product and a proper combination of phage and bacterial attachment sites, a large pool of λ DNA and some activity of the excision gene xis are present. In addition, turnoff of both phage DNA synthesis and xis gene activity are required.     

Cloning of the yeast ribosomal DNA repeat unit in SstI and HindIII lambda vectors using genetic and physical size selections.

The size of DNA fragments complementary to ribosomal RNA was determined in SstI and HindIII restriction spectra from totally and partially cleaved yeast (Saccharomyces cerevisiae) DNA. The results indicated that the yeast ribosomal RNA gene cluster consists of 9000 base-pair long tandemly repeated units. Three different repeating units, which are overlapping with respect to their sequences, were cloned as SstI and HindIII fragments with λ vectors. The isolation of these clones was facilitated by genetic or physical preselection for those recombinant phage which contained DNA inserts in the expected size range. Both preselection methods gave about a 30-fold purification with respect to the λ-rDNA clones. A heteroduplex analysis of the clones obtained with a three-component HindIII vector showed that the center part of the λ genome carrying λ recombination and regulation genes (57 to 77% λ) can become inverted without apparent decrease of growth capacities.     

Characterization of the deoxyribonuclease determined by lambda reverse as exonuclease VIII of Escherichia coli.

Gottesman et al. (1974) detected a new DNAase in Escherichia coli infected with λ reverse, a recombination-proficient substitution mutant of phage λ which is deleted for the λ recombination genes. We have purified this enzyme, using the procedure developed for the purification of exonuclease VIII (Kushner et al., 1974), a DNAase produced by E. coli K-12 strains carrying sbcA^? mutations. The λ reverse exonuclease (Exoλrev) is identical to exonuclease VIII by several criteria. The two enzymes elute at similar salt concentrations from DEAE-cellulose and DNA-cellulose; sediment at the same velocity in glycerol gradients, corresponding to a molecular weight of about 1.4 × 10⁵; migrate at the same R_F in sodium dodecyl sulfate/polyacrylamide gels, indicating a polypeptide molecular weight of 1.4 × 10⁵; exhibit maximum activity at 20 mm-Mg²⁺ and pH 8 to 9; and are much more active on double-stranded DNA than on heat-denatured DNA. Both enzymes are rendered sedimentable by antiserum against Exoλrev. This evidence supports the hypothesis that the non-λ DNA substitution in λ reverse includes recE, the structural gene for exonuclease VIII.     

Properties of a mutant of Escherichia coli defective in bacteriophage lambda head formation (groE). I. Initial characterization

Three mutant strains of Escherichia coli were independently isolated based upon their inability to propagate bacteriophage λ. The strain most extensively studied, NS-1, has a pleiotropic temperature sensitive alteration that affects cell growth, stable RNA synthesis and λ propagation. Labeling experiments and colorimetric determinations of total RNA carried out in this strain demonstrate that within the first five minutes after raising the temperature to 44.5 °C the rate of total RNA accumulation is reduced to a level that is about 15% that of the control, while protein and DNA synthesis continue at nearly normal rates for at least 30 min. This effect is either due to a very rapid degradation of stable RNA species or a reduced synthesis of RNA. Although the accumulation of all stable RNA species (23, 16 and 4 S RNAs) is reduced co-ordinately to levels ranging from 12 to 16% that of the control, the synthesis of messenger RNA is affected to a lesser degree, if at all. The defect in RNA accumulation can be partially reversed by the addition of chloramphenicol at the moment of temperature shift.In addition to phage λ these strains are unable to propagate RNA phage R17 and lambdoid phages φ80, 21 and 434 at elevated temperatures. The growth of phages T4, T7, P1 and P2 is normal.A genetic analysis of strain NS-1 indicates that all of its temperature sensitive properties depend on a mutation, designated groE-1, which co-transduces with a mel (melibiose) marker. However, the expression of the RNA synthesis defect requires, in addition, a second mutation which does not co-transduce with mel.     

On the lack of host-cell reactivation of UV-irradiated phage T5. I. Interference of T5 infection with the host-cell reactivation of phage T1

UV-irradiated phage T5, in contrast to T1, T3 and T7, fail to display hostcell reactivation (HCR) when infecting excision-repair proficient Escherichia coli cells. Possible causes of this lack of HCR (which T5 shares with the T-even phages) have been investigated by studying HCR of T1 under conditions of superinfection by T5. Repair-proficient B/r cells were infected at low multiplicity with UV-irradiated phage T1 in the presence of 1.8 mg/ml caffeine and were superinfected after 15 min with heavily UV-irradiated T5 amber mutants at high multiplicity. The caffeine, which is later diluted out, prevents any T1 repair prior to T5 superinfection, and UV (254 nm) irradiation of T5 with 144 J/m² reduces the ability of this phage to exclude T1, thus permitting a reasonable fraction of the mixedly infected complexes to produce T1 progeny.Under these conditions, T5 superinfection causes loss of HCR in about 90% of the T1-producing complexes. Superinfection with unirradiated T5 likewise inhibits HCR of T1, but superinfection with irradiated T3 (a host-cell-reactivable phage) does not. This indicates that the observed HCR inhibition of T1 results from T5 infection rather than from competition of irradiated foreign DNA for the excision-repair enzymes of the bacterial host. Employment of apropriate T5 amber mutants has shown that “first-step transfer” (FST) of T5 DNA (involving only 8% of the T5 genome) is sufficient for HCR inhibition, but that transfer of the remainder DNA in addition inhibits a previously described minor T1 recovery process. HCR inhibition of T1, and thus presumably lack of HCR in T5 itself, is ascribed to a substance which is produced either post infection by a gene located in the FST segment of the T5 genome, or which is transferred from extracellular T5 together with the FST DNA.     

Physical mapping of the transfer RNA genes on lambda-h80dglytsu+36

The three Escherichia coli transfer RNA genes of the DNA of the transducing phage λ80cI857S^?t68dglyTsu⁺₃₆tyrTthrT (abbreviated λh80T), which specify the structures of tRNA^Gly₂(su⁺₃₆), tRNA^Tyr₂ and tRNA^Thr₃, have been mapped by hybridizing ferritin-labeled E. coli tRNA to heteroduplexes of λh80T DNA with the DNA of the parental phage (λh80cI857S^?t68) and examining the product in the electron microscope. The DNA of λh80T contains a piece of bacterial DNA of length 0·43 λ unit³ that replaces a piece of phage DNA of length 0·46 λ unit, proceeding left from B · P′ (the junction of bacterial DNA and phage DNA) (i.e. att⁸⁰). A cluster of three ferritin binding sites, and thus of tRNA genes, is seen at a position of 0·24 λ unit (1·1 × 10⁴ nucleotides) to the left of B· P′. The three tRNA genes of the cluster are separated by the unequal spacings of 260 (±30) and 140 (± 30) nucleotides, proceeding left from B·P′. The specific map positions have been identified by hybridization competition between ferritin-labeled whole E. coli tRNA with unlabeled purified tRNA^Tyr₂ and with unlabeled partially purified tRNA^Gly₂. The central gene of the cluster is tRNA^Tyr₂. The tRNA^Gly₂gene is probably the one furthest from B·P′. Thus, the gene order and spacings, proceeding left from B·P′, are: tRNA^Thr₃, 260 nucleotides, tRNA^Try₂, 140 nucleotides, tRNA^Gly₂.     

Packaging of coliphage lambda DNA. II. The role of the gene D protein

The gene D protein (pD) of coliphage λ is normally an essential component of the virus capsid. It acts during packaging of concatemeric λ DNA into the phage prohead and is necessary for cutting the concatemers at the cohesive end site (cos). In this report we show that cos cutting and phage production occur without pD in λ deletion mutants whose DNA content is less than 82% that of λ wild type. D-independence appears to result directly from DNA loss rather than from inactivation (or activation) of a phage gene. (1) In cells mixedly infected with undeleted λ and a deletion mutant, particles of the deletion mutant alone are efficiently produced in the absence of pD; and (2) D-independence cannot be attributed to loss of a specific segment of the phage genome. pD-deficient phage resemble pD-containing phage in head size and DNA ends; they differ in their extreme sensitivity to EDTA, greater density, and ability to accept pD.pD appears to act by stabilizing the head against disruption by overfilling with DNA rather than by changing the capacity of the head for DNA. This is shown by the observation that the amount of DNA packaged by a “headful” mechanism, normally in excess of the wild-type chromosome size, is not reduced in the absence of pD. In fact, pD is required for packaging headfuls of DNA. This implies that a mechanism exists for preventing the entry of excess DNA into the head during packaging of concatemers formed by deletion mutants, and we suggest that this is accomplished by binding of cos sites to the head.The above results show that pD is not an essential component of the nuclease that cuts λ concatemers at cos during packaging, and they imply that 82% of a wild-type chromosome length can enter the prohead in the absence of pD. Yet, pD is needed for the formation of cohesive ends after infection with undeleted phage. We propose two models to account for these observations. In the first, cos cutting is assumed to occur early during packaging. The absence of pD leads to release of packaged DNA and the loss of cohesive ends by end-joining. In the second, cos cutting is assumed to occur as a terminal event in packaging. pD promotes cos cutting indirectly through its effect on head stability. We favor the second model because it better explains the asymmetry observed in the packaging of the chromosomes of cos duplication mutants (Emmons, 1974).     

Evidence for sequence preferences in the intercalative binding of ethidium bromide to dinucleoside monophosphates.

The rate of production of tandem duplications in phage λ has been measured in the presence and absence of known recombination systems. Two deletion phages have been used: tdel33, a deletion derivative of a φ80-λ hybrid phage, and λb221, which carries a large deletion of the central portion of the λ chromosome. Both phages are int⁻, and tdel33 is also red⁻, by virtue of their deletions. Stocks of these phages can be prepared free of long tandem duplication derivatives by CsCl density gradient purification. After a single cycle of lytic growth, lysates from these purified phage stocks contain tandem duplications at a frequency of 10⁻³ in the case of tdel33 and 10⁻⁵ in the case of λb221. These frequencies are unaffected by the presence of mutations in the host Rec system or the phage Red system. To investigate the difference in duplication frequency between tdel33 and λb221, the phages were grown in mixed infection. The result indicates that a trans-active product of tdel33 is responsible for its high frequency of duplication production.Tandem duplications have been detected by banding the phage lysates in CsCl density gradients. Long DNA addition mutants can be detected in this way if they arise with a frequency of at least 10⁻⁵ and if the duplication length is at least 0.14 λ lengths. To accomplish this it is necessary to distinguish them from contaminating parental phage and from dense phages with aberrant structures which arise at roughly comparable frequencies. The former can be done by rebanding and the latter by growth and rebanding. To distinguish these types we have also made use of a new mutant of Escherichia coli which does not plate λ deletion phages. All of the DNA addition mutants we have detected in this way are tandem duplications; evidently mutants with long insertions arise more rarely.     

A Mutant of E. COLI That Restricts Growth of Bacteriophage T4 at Elevated Temperatures

After nitrosoguanidine mutagenesis, a Phage Host Defective (phd) mutant of E. coli HfrH was isolated that supported the growth of T4D wild-type bacteriophage at 30°, but not at 40° or higher. Eleven independent spontaneous mutants of T4 (go mutants) were isolated that overcame the growth restriction at high temperature. All of these mutants were located within three percent recombination of a gene 39 amber mutation in the clockwise direction on the standard map. In mixed infections, the representative go mutant chosen for further study seems to be recessive to its wild-type allele. Temperature-shift experiments suggested that the mutated host function involved in phage growth is a "late" function, beginning in mid-eclipse.—Electrophoresis of phage proteins labelled early and late in infection showed that under restrictive conditions early protein synthesis was normal, but that certain late proteins were absent. However, measurements of DNA synthesis showed that under restrictive conditions the amount of phage DNA synthesized, and especially the amount of DNA sedimenting as high molecular weight replicative intermediate, was reduced. Pulse-chase experiments showed that the phage DNA made under restrictive conditions was not rapidly degraded.     

Rec-mediated recombinational hot spot activity in bacteriophage lambda. III. Chi mutations are site-mutations stimulating rec-mediated recombination.

Lambda phage defective for Red and gam function make small plaques on rec⁺ bacteria. Mutants (called Chi) of λ arise which suppress the small-plaque phenotype. Chi mutations arise at at least four well-separated sites; one site is between gene L and att, one is between att and gam, one is in the cII gene, and one is near gene S. A phage strain carrying a Chi mutation at a given site has an extraordinarily high rate of Rec-mediated crossing-over near that site.     

Initiation of recA+-dependent recombination in Escherichia coli (lambda). II. Specificity in the induction of recombination and strand cutting in undamaged covalent circular bacteriophage 186 and lambda DNA molecules in phage-infected cells.

Covalent circular λ DNA molecules produced in Escherichia coli (λ) host cells by infection with labeled λ bacteriophages are cut following superinfection with λ phages damaged by exposure to psoralen and 360 nm light. This cutting of undamaged covalent circular molecules is referred to as “cutting in trans”, and could be a step in damage-induced recombination (Ross &; Howard-Flanders, 1977). Similar experiments performed with the temperate phage 186, which is not homologous with phage λ, showed cutting in trans and damage-induced recombination to occur in homoimmune crosses with phage 186 also. Double lysogens carrying both λ and 186 prophages were used in a test for specificity in cutting in trans and in damage-induced recombination. The double lysogens were infected with ³H-labeled 186 and ³²P-labeled λ phages. When these doubly infected lysogens containing covalent circular phage DNA molecules of both types were superinfected with psoralen-damaged 186 phages and incubated, the covalent circular 186 DNA was cut, while λ DNA remained intact. Similarly, superinfection with damaged λ phages caused λ, but not 186, DNA to be cut. Evidently, cutting in trans was specific to the covalent circular DNA homologous to the DNA of the damaged phages. Homoimmune phage-prophage genetic crosses were performed in the double lysogenic host infected with genetically marked λ and 186 phages. Damage-induced recombination was observed in this system only between the damaged phage DNA and the homologous prophage, none being detected between other homolog pairs present in the same cell. This result makes it unlikely that the damaged phage DNA induces a general state of enhanced strand cutting and genetic recombination affecting all homolog pairs present in the host cell. The simplest interpretation of the specificity in cutting and in recombination is as follows. When they have been incised, the damaged phage DNA molecules are able to pair directly with their undamaged covalent circular homologs. The latter molecules are cut in a recA ⁺ -dependent reaction by a recombination endonuclease that cuts the intact member of the paired homologs.     

Mechanism of potent mutagenic action of N-methyl-N'-nitro-N-nitrosoguanidine on intracellular phage lambda.

N-Methyl-N′-nitro-N-nitrosoguanidine efficiently induces mutations from “clear” to “virulent” in phage λ only during the intracellular growth phase. Lambda DNA extracted from infected bacteria after treatment with MNNG³ produced a mutant yield about 100-fold higher than the spontaneous level upon transfection of MNNG-treated spheroplast cells, whereas the yield diminished an order of magnitude when assayed on untreated spheroplasts. As measured by ¹⁴C incorporation after treatment with [methyl-¹⁴C]MNNG, λ DNA packed in head protein was methylated to about 3% by an MNNG dose of 0.6 mg/ml but was barely mutagenised; whereas intracellular λ DNA was methylated to no more than 0.6% by an MNNG dose of 0.09 mg/ml and was highly mutagenised. Lambda phages treated in vitro with ethyl methanesulfonate produced a rather low mutant yield on untreated cells but the yield increased about tenfold on MNNG-treated cells. Mutability of untreated λ on cells having received an F′ factor was enhanced efficiently by ultraviolet light, but not so by MNNG, previously applied to the F′. Surprisingly similar MNNG dose-effect curves exist for enhancing spontaneous, mispairing (MNNG or EMS induced) and misrepair (ultraviolet light induced) mutagenesis of λ. From these and other data we conclude that MNNG hypermutagenesis results from a synergistic increase in mispairing probability of appropriately methylated bases (by action of MNNG in vivo) in the target gene within an MNNG-induced intracellular environment that has an enhanced mutagenic capacity.