Electron microscopy study of viral DNA packaging with lambda head mutants

In a previous study, various intermediates in λ DNA packaging were visualized after lysis of λ-infected cells with osmotic shock and sedimentation through a sucrose formalin cushion onto electron microscope grids. Along this line, a systematic screening for intermediates accumulated in all head mutants available was performed. λA^?-infected cells accumulate only empty spherical protein shells (petit λ) bound at an intermediate point along the DNA thread. In situ digestion experiments with restriction endonuclease EcoRI show that the petit λ-DNA complexes are formed at a fixed point on the DNA concatemer. In $\text{λNu1}{}^{\mathrm{?}}\text{-}\text{infected}$ cells, however, most petit λ was not bound to DNA. In Fec^? cells, which are defective in formation of concatemers but normal in head protein synthesis, most petit λ did not sediment onto the carbon film of the grid. In $\text{D}{}^{\mathrm{?}}$ mutant, petit λ, partially full heads and empty heads with released DNA were observed. λFI^?-infected cells also accumulate petit λ and partially full heads. The present studies suggest that protein pNu1 is required for complex formation between head precursors and DNA concatemers, $\text{p}\text{A}$ for the initiation of DNA packaging, $\text{p}\text{D}$ and $\text{p}\text{F}$I for the promotion of DNA packaging, and $\text{p}\text{D}$ for stabilization of head structures. The results obtained with other head mutants involved in formation of mature proheads and head completion confirm earlier results obtained by different techniques.     

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.     

Studies related to the head-maturation pathway of bacteriophages T4 and T2: I. Morphology and kinetics of intracellular particles produced by mutants in the maturation genes

Mutants in the genes governing the maturation of the head of bacteriophage T4 and in gene 24 were studied by electron microscopy of thin sections. We define morphologically: black particles, comprising mature, stable heads and immature, fragile heads, which break down upon lysis; grizzled particles, which apparently are partially filled or partially emptied; empty large particles without DNA or core Which are all the same size as normal heads; empty small particles without DNA and without core which are of the size of the τ particle, which is the prehead of phage T4. The study of single and double mutants of the maturation genes demonstrates that the phenotypes are only different by the proportions of the different particles made except for 17^? where only empty small and empty large particles accumulate. The mutants in gene 24 are epistatic on all other mutants. Mutants in gene 17 are epistatic on the remaining ones. The results are consistent with the hypothesis that the products of several of the maturation genes act on DNA to render it competent for packaging while the others act directly on the particle. By this uncoupling, bypasses and abortive pathways can result.     

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.     

Assembly of bacteriophage lambda heads: Protein processing and its genetic control in petit λ assembly

Petit bacteriophage λ is a hollow λ head precursor which is found in λ-infected lysates, including lysates of phage λ carrying mutations in head genes. Wild-type petit λ has a protein composition similar to heads, except that it is missing pD ⁴, a major component of heads. About 95% of the mass of petit λ is pE, the major structural protein of heads, and in addition it has proteins pB, h3, X1, and X2. Tryptic fingerprint analysis shows that h3 is a proteolytic cleavage product of pB, and previous experiments have shown that X1 and X2 are protein fusion products, closely related to each other and containing amino acid sequences of both pC and pE. Petit lambdas derived from infection by phages defective in genes A or D are indistinguishable from wild-type petit λ. B⁻, C⁻, or groE⁻ defective petit lambdas show differences from wild-type in protein composition and in extent of protein processing. On the basis of the properties of mutant petit lambdas it is concluded that: (1) the protein processing reactions (cleavage of pB; fusion of pC with pE) occur on the petit λ structure; (2) cleavage of pB requires the functioning of genes C and groE but not A or D; (3) fusion of pC and pE requires gene groE but not A, B or D; (4) pNu3 participates directly in petit λ assembly but is lost from the structure by the time assembly is complete.Physical studies of petit λ show that wild-type, A⁻, B⁻ and D⁻ petit lambdas sediment at 150 S, while C⁻ and groE⁻ petit lambdas sediment at 190 S. Purified petit λ of either class has an ultraviolet absorption spectrum characteristic of pure protein.     

Studies of DNA packaging into the heads of bacteriophage lambda

During the assembly of bacteriophage λ heads, a head-like, DNA-free structure called petite λ, is first constructed. Into this, λ DNA is then packaged. In this paper we examine early interactions between λ DNA and petite λ in a cell-free system. The two major findings of this paper are: (1) when seen through the electron microscope, an early petite λ-λ DNA complex appears with the circular petite λ having the DNA crossing through its center. These resemble a bead on a string or the Greek letter φ (hence they are called φ structures). The λ A protein is required in the formation of φ structures. Also, φ structures can be found in bacteria infected with phage λ. (2) The polyamine putrescine is required for phage head assembly. An earlier reported requirement for spermidine can be replaced by the addition of putrescine. Polyamine is required in the DNA packaging reaction after the packaging has begun.     

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.     

Construction and analysis of a genetically tuneable lytic phage display system

The Bacteriophage λ capsid protein gpD has been used extensively for fusion polypeptides that can be expressed from plasmids in Escherichia coli and remain soluble. In this study, a genetically controlled dual expression system for the display of enhanced green fluorescent protein (eGFP) was developed and characterized. Wild-type D protein (gpD) expression is encoded by λ Dam15 infecting phage particles, which can only produce a functional gpD protein when translated in amber suppressor strains of E. coli in the absence of complementing gpD from a plasmid. However, the isogenic suppressors vary dramatically in their ability to restore functional packaging to λDam15, imparting the first dimension of decorative control. In combination, the D-fusion protein, gpD::eGFP, was supplied in trans from a multicopy temperature-inducible expression plasmid, influencing D::eGFP expression and hence the availability of gpD::eGFP to complement for the Dam15 mutation and decorate viable phage progeny. Despite being the worst suppressor, maximal incorporation of gpD::eGFP into the λDam15 phage capsid was imparted by the SupD strain, conferring a gpDQ68S substitution, induced for plasmid expression of pD::eGFP. Differences in size, fluorescence and absolute protein decoration between phage preparations could be achieved by varying the temperature of and the suppressor host carrying the pD::eGFP plasmid. The effective preparation with these two variables provides a simple means by which to manage fusion decoration on the surface of phage λ.     

Crystal structure of the coat protein of the flexible filamentous papaya mosaic virus

The terminase motors of bacteriophages have been shown to be among the strongest active machines in the biomolecular world, being able to package several tens of kilobase pairs of viral genome into a capsid within minutes. Yet, these motors are hindered at the end of the packaging process by the progressive buildup of a force-resisting packaging associated with already packaged DNA. In this experimental work, we raise the issue of what sets the upper limit on the length of the genome that can be packaged by the terminase motor of phage λ and still yield infectious virions and the conditions under which this can be efficiently performed. Using a packaging strategy developed in our laboratory of building phage λ from scratch, together with plaque assay monitoring, we have been able to show that the terminase motor of phage λ is able to produce infectious particles with up to 110% of the wild-type λ-DNA length. However, the phage production rate, and thus the infectivity, decreased exponentially with increasing DNA length and was a factor of 10(3) lower for the 110% λ-DNA phage. Interestingly, our in vitro strategy was still efficient in fully packaging phages with DNA lengths as high as 114% of the wild-type length, but these viruses were unable to infect bacterial cells efficiently. Further, we demonstrated that the phage production rate is modulated by the presence of multivalent ionic species. The biological consequences of these findings are discussed.     

Isolation and structure of phage lambda head-mutant DNA

High molecular weight DNA accumulates in bacteria in which λ is multiplying but cannot complete the formation of new phage particles due to a defect in head assembly. Accumulated λ DNA has been isolated from induced mitomycin C-treated lysogens by means of a shift in buoyant density labels from heavy to light and fractionation by density-gradient sedimentation for completely light DNA. Head formation was blocked in these lysogens by amber mutations in genes D or E, which specify the two major head proteins. The purified DNA is at least 80% λ by DNA-DNA hybridization and some preparations are close to 100% λ by this test.     

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.     

Virus DNA packaging: the strategy used by phage λ

Phage λ, like a number of other large DNA bacterio-phages and the herpesviruses, produces concatemeric DNA during DNA replication. The concatemeric DNA is processed to produce unit-length, virion DNA by cutting at specific sites along the concatemer. DNA cutting is coordinated with DNA packaging, the process of translocation of the cut DNA into the preformed capsid precursor, the prohead. A key player in the λ DNA packaging process is the phage-encoded enzyme terminase, which is involved in (i) recognition of the concatemeric λ DNA; (ii) initiation of packaging, which includes the introduction of staggered nicks at cosN to generate the cohesive ends of virion DNA and the binding of the prohead; (iii) DNA packaging, possibly including the ATP-driven DNA translocation; and (iv) following translocation, the cutting of the terminal cosN lo complete DNA packaging. To one side of cosN is the site cosB, which plays a role in the initiation of packaging; along with ATP, cosB stimulates the efficiency and adds fidelity to the endo-nuclease activity of terminase in cutting cosN. cosB is essential for the formation of a post-cleavage complex with terminase, complex I, that binds the prohead, forming a ternary assembly, complex II. Terminase interacts with cosN through its large subunit, gpA, and the small terminase subunit, gpNul, interacts with cosB. Packaging follows complex II formation. cosN is flanked on the other side by the site cosQ, which is needed for termination, but not initiation, of DNA packaging. cosQ is required for cutting of the second cosN, i.e. the cosN at which termination occurs. DNA packaging in λ has aspects that differ from other λ DNA transactions. Unlike the site-specific recombination system of λ, for DNA packaging the initial site-specific protein assemblage gives way to a mobile, translocating complete, and unlike the DNA replication system of λ, the same protein machinery is used for both initiation and translocation during λ DNA packaging.     

Testing models of the arrangement of DNA inside bacteriophage lambda by crosslinking the packaged DNA

Bis-psoralens can make crosslinks between two adjacent segments of a condensed DNA molecule. We have used bis-psoralen crosslinking as a covalent means of preserving structural features of DNA packaged inside bacteriophage λ. A single bis-crosslink prevents normal electron microscopic spreading of intact λ DNA: after deproteinization the molecules appear as tangled rosettes which are presumably due either to trapped knots or supercoils. However, restriction nuclease digestion of the crosslinked DNA yields fragments that spread normally. The location of crosslinks can be studied by their appearance in such a digest as X-shaped molecular features. Significant crosslinking frequencies are found between all six possible pairs of the four largest BglII fragments of λ DNA. Little or no evidence is seen for crosslinked loops within individual fragments. These results are inconsistent with two previously suggested models of intraphage DNA packaging. Determination of the positions of crosslinks within restriction fragments yields a pattern of DNA contacts too complex for any simple analysis. The finding of hints of periodicity in the sites of crosslinks, preferential crosslinking of some restriction fragments, and the occurrence of one particularly efficient crosslinking reaction between two restriction fragments appear to rule out purely random packaging arrangements.     

Bacteriophage lambda FII gene protein: role in head assembly

The in vitro completion of bacteriophage lambda F_II^? heads to form phage can be used as an assay for the λ F_II gene protein. F_II protein activity is released from highly purified phage particles or phage heads by treatment with heat or denaturing agents. F_II protein was purified from isolated phage particles and from an extract of E^? infected cells in which it is not bound to any large structures. No differences in molecular weight (11,500), isoelectric point (4.75), electrophoretic mobility, or purification properties could be demonstrated between the F_II proteins from the two sources. Thus the polypeptide does not seem to be modified during assembly.Phage φ80 is closely related to λ. φ80 heads will join to φ80 tails in vitro but will not join to λ tails, though λ heads will join to either type of tail. Mixing experiments between F_II^? heads, tails, and F_II protein from λ or φ80 show that the specificity of head-tail joining is correlated with the source of the F_II protein and not with the source of the other head proteins. Thus, F_II protein is apparently responsible for this specificity of head-tail joining.     

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.     

Efficiency and fidelity of human DNA polymerases λ and β during gap-filling DNA synthesis

The base excision repair (BER) pathway coordinates the replacement of 1-10 nucleotides at sites of single-base lesions. This process generates DNA substrates with various gap sizes which can alter the catalytic efficiency and fidelity of a DNA polymerase during gap-filling DNA synthesis. Here, we quantitatively determined the substrate specificity and base substitution fidelity of human DNA polymerase λ (Pol λ), an enzyme proposed to support the known BER DNA polymerase β (Pol β), as it filled 1-10-nucleotide gaps at 1-nucleotide intervals. Pol λ incorporated a correct nucleotide with relatively high efficiency until the gap size exceeded 9 nucleotides. Unlike Pol λ, Pol β did not have an absolute threshold on gap size as the catalytic efficiency for a correct dNTP gradually decreased as the gap size increased from 2 to 10 nucleotides and then recovered for non-gapped DNA. Surprisingly, an increase in gap size resulted in lower polymerase fidelity for Pol λ, and this downregulation of fidelity was controlled by its non-enzymatic N-terminal domains. Overall, Pol λ was up to 160-fold more error-prone than Pol β, thereby suggesting Pol λ would be more mutagenic during long gap-filling DNA synthesis. In addition, dCTP was the preferred misincorporation for Pol λ and its N-terminal domain truncation mutants. This nucleotide preference was shown to be dependent upon the identity of the adjacent 5'-template base. Our results suggested that both Pol λ and Pol β would catalyze nucleotide incorporation with the highest combination of efficiency and accuracy when the DNA substrate contains a single-nucleotide gap. Thus, Pol λ, like Pol β, is better suited to catalyze gap-filling DNA synthesis during short-patch BER in vivo, although, Pol λ may play a role in long-patch BER.     

In vivo isolation of a pPJ3a-lambda cosmid mediated by the Tn2301 transposon

From an E. coli cell harboring plasmid pPJ3b (= pPJ3a::Tn2301) and infected with phage λ, we have isolated two defective phages having inserted pPJ3a DNA and Tn2301 in their genomes. One of them has been extensively characterized: it behaves like a cosmid, i.e., upon injection into the cell, its DNA circularizes and replicates as a plasmid (pPJ10); it can be packaged again in λ heads, provided the presence of a phage helper. Furthermore, heteroduplex analysis has shown that in pPJ10, the transposon Tn2301 is inverted compared to its direction in pPJ3b. We give evidence suggesting that this type of inversion is in part mediated by Tn2301.     

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).     

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.