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).     

Processing of bacteriophage lambda DNA during its assembly into heads

DNA purified from bacteriophage λ added to a cell-free extract derived from induced λ lysogens can be packaged into infectious phage particles (Kaiser & Masuda, 1973). In this paper the structure of the DNA which is the substrate for in vitro packaging and head assembly is described. The active precursor is a multichromosomal polymer that contains covalently closed cohesive end sites. Neither circular or linear DNA monomers nor polymers with unsealed cohesive ends are packaged efficiently into heads. The unit length monomer is packaged when it is either contained in the interior of a polymer (both of its ends are in cos sites) or when it has a free left end and a cos site on its right. The monomer unit with a free right end is not a substrate for packaging.A procedure is given for the purification of λ DNA fragments that contain either the left or the right cohesive end. The fragments are produced by digesting λ DNA with the site-specific Escherichia coli R₁ endonuclease; the left and right ends are separated by sedimentation through a sucrose gradient. These fragments are used to construct small polymers that have a unit length λ monomer with (1) a free left end and a closed right end, (2) a free right end and a closed left end, or (3) both ends closed in cos sites.     

Bacteriophage lambda derivatives carrying two copies of the cohesive end site

A spontaneously arising tandem duplication derivative of bacteriophage lambda has been isolated, which carries two copies of the site where the cohesive ends are formed (designated cos). Its structure has been determined by electron microscopy of DNA heteroduplexes. These heteroduplexes reveal that the duplication is usually, but not always, carried on the left end of the chromosome. A second duplication phage having two copies of cos, constructed by Feiss &; Campbell (1974), has also been studied by electron microscopy and is found to have a similar property.Unlike most tandem duplication derivatives of phage λ, the mutant studied here is not stable during growth in the absence of generalized recombination, but segregates both the triplication and the parental phage. This verifies that both cos sites are functional. The triplication does not arise as a result of end-to-end aggregation of phage chromosomes or site-specific recombination catalyzed by the chromosome maturation system at cos. It must therefore result from the cutting of mature ι chromosomes from concatemeric replication intermediates. The pattern of cutting observed shows that the λ cohesive ends are not created by a free nuclease acting on unpackaged DNA. The cutting appears to be influenced by the amount of DNA previously packaged into a phage head. A model for λ packaging is presented which explains the results.The duplication phage of Feiss &; Campbell (1974) carries a novel addition containing self-complementary sequences.     

Polarized packaging of bacteriophage lambda chromosomes.

Packaging of chromosomes during lytic growth of cohesive end-site (cos site) duplication strains of phage lambda is strikingly asymmetric; the duplication segment is generally at the left chromosome end (Emmons, 1974). In the present study, the packaging of non-replicating cos duplication chromosomes is shown to be similarly asymmetric. It is, therefore, likely that the packaging process itself is polarized, in an A to R direction. This conclusion is based on the study of packaging of repressed prophage chromosomes of dilysogenic strains of Escherichia coli by a heteroimmune helper. In these strains one of the two prophages contains a cos duplication (see Fig. 2). The frequency with which helper-packaged chromosomes carry the cos duplication segment agrees well with expectations derived from lytically grown phage.Haploid segregants are produced from the cos duplication strain at a lower level (35%) during lytic growth than during packaging of repressed prophage chromosomes (50%). This is expected if chromosomes are packaged processively (in sequence) during lytic growth.Packaging of repressed cos triplication chromosomes by a heteroimmune helper also yields a distribution of haploid and duplication chromosomes that agrees with expectations from lytically grown cos duplication phage and the assumption that the initial cutting of a cos site to initiate a packaging sequence is made at random.Polarized, processive packaging and random initial cutting are elements of a model of lambda chromosome packaging proposed by Emmons (1974), for which our experiments provide support.     

Sequencing and analysis of the cos region of the lactococcal bacteriophage c2

The cohesive termini of the DNA genome of the lactococcal bacteriophage c2 were directly sequenced and appeared to be complementary, non-symmetrical, 9-nucleotide single-stranded, 3′ extended DNAs, with the following sequence: 5′-GTTAGGCTT-3′ 3′-CAATCCGAA-5′. DNA located on either side of the cohesive ends was sequenced and several repeats and a region with the potential for a DNA bend were found. Previously sequenced cos regions of 13 other bacteriophages were also examined for similar sequence features. All of the bacteriophages from gram-positive hosts had 3′ extended DNA termini, in contrast to the bacteriophages from gram-negative hosts, which had 5′ extended DNA termini. All bacteriophages had a region of dyad symmetry close to the cohesive termini. A 7.3 kb DNA fragment of the c2 genome containing the cos sequences was cloned; transduction experiments demonstrated that these cloned sequences could act as a substrate for packaging enzymes of phage c2.     

DNA Packaging Specificity of Bacteriophage N15 with an Excursion into the Genetics of a Cohesive End Mismatch

During DNA replication by the λ-like bacteriophages, immature concatemeric DNA is produced by rolling circle replication. The concatemers are processed into mature chromosomes with cohesive ends, and packaged into prohead shells, during virion assembly. Cohesive ends are generated by the viral enzyme terminase, which introduces staggered nicks at cos, an approx. 200 bp-long sequence containing subsites cosQ, cosN and cosB. Interactions of cos subsites of immature concatemeric DNA with terminase orchestrate DNA processing and packaging. To initiate DNA packaging, terminase interacts with cosB and nicks cosN. The cohesive ends of N15 DNA differ from those of λ at 2/12 positions. Genetic experiments show that phages with chromosomes containing mismatched cohesive ends are functional. In at least some infections, the cohesive end mismatch persists through cyclization and replication, so that progeny phages of both allelic types are produced in the infected cell. N15 possesses an asymmetric packaging specificity: N15 DNA is not packaged by phages λ or 21, but surprisingly, N15-specific terminase packages λ DNA. Implications for genetic interactions among λ-like bacteriophages are discussed.     

EcoRI analysis of bacteriophage P22 DNA packaging.

Bacteriophage P22 linear DNA molecules are a set of circularly permuted sequences with ends located in a limited region of the physical map. This mature form of the viral chromosome is cut in headful lengths from a concatemeric precursor during DNA encapsulation. Packaging of P22 DNA begins at a specific site, which we have termed pac, and then proceeds sequentially to cut lengths of DNA slightly longer than one complete set of P22 genes (Tye et al., 1974b). The sites of DNA maturation events have been located on the physical map of EcoRI cleavage sites in P22 DNA. EcoRI digestion products of mature P22 wild-type DNA were compared with EcoRI fragments of two deletion and two insertion mutant DNAs. These mutations decrease or increase the length of the genome, but do not alter the DNA encapsulation mechanism. Thus the position of mature molecular ends relative to EcoRI restriction sites is different in each mutant, and comparison of the digests shows which fragments come from the ends of linear molecules. From the positions of the ends of molecules processed in sequential headfuls, the location of pac and the direction of encapsulation relative to the P22 map were deduced. The pac site lies in EcoRI fragment A, 4.1 × 10³ base-pairs from EcoRI cleavage site 1. Sequential packaging of the concatemer is initiated at pac and proceeds in the counterclockwise direction relative to the circular map of P22. One-third of the linears in a population are cut from the concatemer at pac, and most packaging sequences do not extend beyond four headfuls.Fragment D is produced by EcoRI cleavage at a site near the end of a linear chromosome which has been encapsulated starting at pac. The position of the pac site is therefore defined by one end of fragment D. The pac site is not located near genes 12 and 18, the only known site for initiation of P22 DNA replication, but lies among late genes at a position on the physical gene map approximately analogous to the cohesive end site (cos) of bacteriophage λ at which λ DNA is cleaved during encapsulation. Our results suggest that P22 and λ DNA maturation mechanisms have many common properties.     

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.     

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.     

Circular Permutation of the Genome of a Temperate Bacteriophage from Streptococcus cremoris BK5

The temperate bacteriophage BK5-T was isolated from Streptococcus cremoris BK5 by induction with mitomycin C. Electron microscopy revealed that BK5-T DNA consists of linear molecules, ranging in size from 39.7 to 46 kilobase pairs. Restriction analysis of self-ligated BK5-T DNA showed that the ends of the DNA were not cohesive. The EcoRI restriction fragments of the phage genome were cloned into pACYC184. Restriction enzyme analysis of both the phage DNA and the cloned EcoRI fragments with EcoRI, BstEII, PstI, ClaI, and XbaI yielded a 37.6-kilobase-pair-long circular restriction map for the phage genome. It was concluded that the BK5-T DNA molecules in the population differ in their sequence by a circular permutation and that individual DNA molecules are terminally redundant. The map location of the sites at which packaging of BK5-T DNA into phage heads is initiated (pac) and at which the phage integrates into the bacterial chromosome (att) were established.     

Single-Molecule and FRET Fluorescence Correlation Spectroscopy Analyses of Phage DNA Packaging: Colocalization of Packaged Phage T4 DNA Ends within the Capsid

Linear DNAs of any sequence can be packaged into empty viral procapsids by the phage T4 terminase with high efficiency in vitro. Packaging substrates of 5 kbp and 50 kbp, terminated by energy transfer dye pairs, were constructed from plasmid and λ phage DNAs. Nuclease and fluorescence correlation spectroscopy (FCS) assays showed that ∼ 20% of the substrate DNA was packaged and that the DNA dye ends of the packaged DNA were protected from nuclease digestion. Upon packaging, both 5-kbp and  50-kbp DNAs produced comparable fluorescence resonance energy transfer (FRET) between Cy5 and Cy5.5 double-dye terminated DNAs. Single-molecule FRET (sm-FRET) and photobleaching analysis shows that FRET is intramolecular rather than intermolecular upon packaging of most procapsids and demonstrates that single-molecule detection allows mechanistic analysis of packaging in vitro. FRET-FCS and sm-FRET measurements are comparable and show that both the 5-kbp and the  50-kbp packaged DNA ends are held within 8-9 nm of each other, within the dimensions of the long axis of the procapsid portal. The calculated distribution of FRET distances is relatively narrow for both FRET-FCS and sm-FRET, suggesting that the two packaged DNA ends are held at the same fixed distance relative to each other in most capsids. Because one DNA end is known to be positioned for ejection through the portal, it can be inferred that both DNAs ends are held in proximity to the portal entrance and ejection channel. The analysis suggests that a DNA loop, rather than a DNA end, is translocated by the packaging motor to fill the procapsid.     

Genetic Variation of Lactobacillus delbrueckii subsp. lactis Bacteriophages Isolated from Cheese Processing Plants in Finland

The genomes of four Lactobacillus delbrueckii subsp. lactis bacteriophages were characterized by restriction endonuclease mapping, Southern hybridization, and heteroduplex analysis. The phages were isolated from different cheese processing plants in Finland between 1950 and 1972. All four phages had a small isometric head and a long noncontractile tail. Two different types of genome (double-stranded DNA) organization existed among the different phages, the pac type and the cos type, corresponding to alternative types of phage DNA packaging. Three phages belonged to the pac type, and a fourth was a cos-type phage. The pac-type phages were genetically closely related. In the genomes of the pac-type phages, three putative insertion/deletions (0.7 to 0.8 kb, 1.0 kb, and 1.5 kb) and one other region (0.9 kb) containing clustered base substitutions were discovered and localized. At the phenotype level, three main differences were observed among the pac-type phages. These concerned two minor structural proteins and the efficiency of phage DNA packaging. The genomes of the pac-type phages showed only weak homology with that of the cos-type phage. Phage-related DNA, probably a defective prophage, was located in the chromosome of the host strain sensitive to the cos-type phage. This DNA exhibited homology under stringent conditions to the pac-type phages.     

Fragmentation of Bacillus bacteriophage phi105 DNA by complementary single-stranded DNA in the cohesive ends of the molecule.

The structure of DNA from the temperate Bacillus subtilis phage phi105 was examined by using the restriction endonuclease EcoRI and by sedimentation analysis. The DNA contains six EcoRI cleavage sites. Although eight DNA fragments were identified in the EcoRI digests, the largest of these was shown to consist of the two fragments that carry the cohesive ends of the phage DNA. In neutral gradients, the majority of whole phi105 DNA sedimented as nicked circles and the remainder as oligomers. No unit-length linear structures were detected. The associated cohesive ends could be sealed by DNA ligase from Escherichia coli and could be cleaved by S1 nuclease. On the basis of these results and previously reported studies, it appears that, as isolated from phage particles, phi105 DNA is a circular molecule that is formed from the linear structure by the association of complementary single-stranded DNA.     

The two-disulphide intermediates and the folding pathway of reduced pancreatic trypsin inhibitor.

We have studied bacteriophage λ head assembly under conditions in which the normal pathways for late phage DNA (concatemer) synthesis are blocked, and early (monomeric circular) DNA replication products accumulate. Our results show that under such conditions, the amount of late protein per amount of DNA is normal, but the amount of phage produced is not. Electron microscopic examination of thin sections of these bacteria shows that large numbers of “empty” head-shaped particles are produced. We conclude that the packaging of λ DNA depends on some structure (or property) possessed by DNA concatemers and absent in monomeric circular molecules and that the empty head-shaped particles which accumulate when concatemer production is blocked are head precursors which would normally accept concatemer DNA.These empty particles are the same size (approximately 550 Å vertex-to-vertex diameter) as the electron-dense, DNA-filled particles observed in similar sections of wild-type infected bacteria. In lysates the empty particles are approximately the same size as they are within the bacteria. However, filled heads observed in thin sections (or in negatively stained preparations) of lysates are larger than they are within the bacteria. This observation is contrary to what was previously suspected, since there seems to be little or no change in the size of intracellular λ capsids as a direct consequence of DNA packaging. Instead, an increase in the size of completed phage heads seems to take place as a consequence of cell lysis.     

Molecular genetic analysis of bacteriophage P22 gene 3 product, a protein involved in the initiation of headful DNA packaging.

Bacteriophage P22 DNA packaging events occur in processive series on concatemeric phage DNA molecules. At the point where such series initiate, the DNA is recognized at a site called pac, and most molecular left ends are generated within six short regions called end sites, which are present in a 120 base-pair region surrounding the pac site. The bacteriophage P22 genes 2 and 3 proteins are required for successful generation of these ends and DNA packaging during progeny virion assembly. Mutants lacking the 162-amino-acid gene 3 protein replicate DNA and assemble functional procapsids. In this report we describe the nucleotide changes and DNA packaging phenotypes of a number of missense mutations of gene 3, which give the phage a higher than normal frequency of generalized transduction. In cells infected by these mutants, more packaging events initiate on the host chromosome than in wild-type infections, so the mutations are thought to affect the specificity of packaging initiation. In addition to having this phenotype, these mutations affect the process of phage DNA packaging in detectable ways. They may: (1) alter the target site specificity for packaging; (2) make target site recognition more promiscuous; (3) affect end site utilization; (4) alter the pac site; and (5) cause apparent random DNA packaging series initiation on phage DNA.     

Physical characterization of bacteriophage MX4, a generalized transducing phage for Myxococcus xanthus.

A generalized transducing bacteriophage of Myxococcus xanthus has been examined. The phage particle consists of an isometric head and a contractile tail. The genome of the phage is a linear DNA molecule of molecular weight 39 ± 2.1 × 10⁶, which contains the normal DNA bases 70% of which are guanosine + cytosine. No overall heterogeneity of base composition is present. The DNA does not carry easily detectable cohesive ends nor is it cyclically permuted. It does contain a large and somewhat variable terminal redundancy. Heating phage particles in the presence of EDTA causes tail sheath contraction and ejection of DNA, some of which remains attached to the tail. Digestion of tail-bound DNA with restriction enzymes shows that the phage tail can be attached to either end of the DNA. Thus the DNA probably contains recognition sites for the packaging of its DNA at both ends. These results suggest possible mechanisms for the genesis of transducing particles by phage MX4.     

Genetic analysis of mutations affecting terminase, the bacteriophage lambda DNA packaging enzyme, that suppress mutations in cosB, the terminase binding site.

Terminase, the DNA packaging enzyme of phage lambda, binds to lambda DNA at a site called cosB, and introduces staggered nicks at an adjacent site, cosN, to generate the cohesive ends of virion lambda DNA molecules. Terminase also is involved in separation of the cohesive ends and in binding the prohead, the empty protein shell into which lambda DNA is packaged. Terminase is a DNA-dependent ATPase, and both subunits, gpNu1 and gpA, have ATPase activity. cosB contains a series of gpNu1 binding sites, R3, R2 and R1; between R3 and R2 is a binding site, I1, for integration host factor (IHF), the Escherichia coli DNA bending protein. In this work, a series of mutations in Nu1 have been isolated as suppressors of cosB mutations. One of the Nu1 mutations is identical to the previously described Nu1ms1/ohm1 mutation predicted to cause the change L40F in the 181 amino acid-long gpNu1. Three other Nu1 missense mutations, the Nu1ms2 (L40I), ms3 (Q97K) and ms4 (A92G) mutations, have been isolated; the relative strengths of suppression of cosB mutations by the Nu1ms mutations are: ms1 > ms2 > ms3 > ms4. The Nu1 missense mutations all affect amino acid residues that lie outside of the putative helix-turn-helix DNA binding motif of gpNu1. The Nu1ms1 and Nu1ms2 mutations alter an amino acid residue (L40) that lies directly between two segments of gpNu1 proposed to be involved in ATP binding and hydrolysis; thus these mutations are likely to alter the gpNu1 ATP-binding site. The Nu1ms3 and Nu1ms4 mutations both affect amino acid residues in the central region of gpNu1 that is predicted to form a hydrophilic alpha-helix. To explain how the Nu1ms mutations suppress cosB defects, models involving alterations of the DNA binding and/or catalytic properties of terminase are considered. The results also indicate that terminase occupancy of a single gpNu1 binding site (R3) is necessary and sufficient for the efficient initiation of DNA packaging; the Nu1ms1, ms2 and ms3 mutations permit IHF-independent plaque formation by a phage lacking R2 and R1.     

Bacteriophage lambda DNA maturation. The functional relationships among the products of genes Nul, A and FI

The FI gene of bacteriophage λ functions in head assembly, but its exact role is not well understood. FI mutants are leaky, producing between 0.1 and 0.5 viable particles per infected cell. In order to investigate the function of the FI product (gpFI) in vivo, mutants of λ were isolated that are able to grow in the absence of gpFI. These mutants, called fin (for FI independence) map in the region of gene Nul and the beginning of gene A.Proteins made in cells infected with the fin mutants were labelled with [³⁵S]methionine and analysed by polyacrylamide gel electrophoresis. In addition, the levels of activity of the A product were measured in the in vitro DNA packaging assay. As a result of these experiments, the fin mutants can be classified in two groups. Upon infection, fin mutants of one group selectively produce three to fivefold more gpA than do wild-type phage fin mutants of the second group do not overproduce any λ late gene product detectable by the autoradiographic technique.gpA overproducers can also be isolated by selecting for λAam Wam phages that can plate on a weak suII cell strain. The mutation responsible for this pseudoreversion is called Aop and maps in the Nu1-A region. Aop is also a fin mutation, since its presence in λFI^? enables it to plate on non-permissive hosts.Therefore, it seems that one condition sufficient for normal growth of FI^? phage is the overproduction of gpA. The nature of the fin mutations that do not result in gpA overproduction is discussed.     

Bidirectional replication from an internal ori site of the linear N15 plasmid prophage

The prophage of coliphage N15 is not integrated into the chromosome but exists as a linear plasmid molecule with covalently closed hairpin ends (telomeres). Upon infection the injected phage DNA circularizes via its cohesive ends. Then, a phage-encoded enzyme, protelomerase, cuts the circle and forms the hairpin telomeres. N15 protelomerase acts as a telomere-resolving enzyme during prophage DNA replication. We characterized the N15 replicon and found that replication of circular N15 miniplasmids requires only the repA gene, which encodes a multidomain protein homologous to replication proteins of bacterial plasmids replicated by a theta-mechanism. Replication of a linear N15 miniplasmid also requires the protelomerase gene and telomere regions. N15 prophage replication is initiated at an internal ori site located within repA and proceeds bidirectionally. Electron microscopy data suggest that after duplication of the left telomere, protelomerase cuts this site generating Y-shaped molecules. Full replication of the molecule and subsequent resolution of the right telomere then results in two linear plasmid molecules. N15 prophage replication thus appears to follow a mechanism that is distinct from that employed by eukaryotic replicons with this type of telomere and suggests the possibility of evolutionarily independent appearances of prokaryotic and eukaryotic replicons with covalently closed telomeres.