Lambda DNA Packaging Research Articles

Probing Functional Motifs and Regions in the Phage Lambda DNA Packaging Motor by Mutagenesis and Single-Molecule Optical Tweezers Measurements

A key step in the assembly of many viruses is the packaging of DNA into preformed procapsids by an ATP-powered molecular motor. To shed light on the motor mechanism we used optical tweezers measurements to study the effect on DNA translocation dynamics of amino acid changes in the large terminase subunit (gpA) of the phage lambda packaging motor. Observed changes in motor velocity, processivity, and/or velocity-force dependence provide evidence supporting the assignment of a Walker A-like phosphate-binding motif, an adenine binding Q motif analogous to that recently identified in RNA helicases, and a C motif that couples ATP hydrolysis to DNA translocation. In addition, we found that a residue change T194M in a predicted loop-helix-loop region outside any previously described motifs, caused a dramatic 8-fold reduction in motor velocity without changing processivity or force-dependence. T194 lies in a loop-helix-loop region that is predicted to position key residues of Walker B and C catalytic motifs and appears to regulate DNA translocation rate in both the lambda DNA packaging motor and the homologous B. Subtilis SpoIIIE chromosome segregation motor.

The bacteriophage lambda DNA packaging enzyme: identification of four structural domains of the gpNu1 subunit using limited proteolysis.

Lambda DNA terminase, the enzyme that cleaves virion-length chromosomes from multigenomic concatemers and packages them into the bacteriophage head, is composed of two subunits, gpNu1 and gpA. Direct determination of the structure of gpNu1, the smaller subunit, has not been possible because of its insolubility in aqueous solutions. Therefore, to identify smaller and potentially water-soluble domains of gpNu1, we analyzed the nature of the products obtained by limited digestion of the protein with several proteases. The gpNu1 subunit was obtained from E. coli cells transfected with the plasmid pH6-Nu1 that overproduces the protein. Incubation of gpNu1 solubized in 2.5 M guanidinium chloride with chymotrypsin resulted in the formation of at least eight discrete protein bands, while treatment with endoproteinase glu-C and bromelain yielded three and one major bands, respectively. The peptides generated by digestion with the various proteases were separated by two-dimensional gel electrophoresis and transferred to Immobilon membranes. Amino acid sequencing of the peptides allowed for the precise assignment of their N-terminal amino acid, while their estimated molecular weights permitted the identification of their C-terminal ends. The results reveal that in the presence of 2.5 M guanidinium chloride, gpNu1 is partially folded in at least four distinct structural domains that correspond to functional domains as determined by previously reported genetic experiments. This information is key to design new plasmids to overproduce these domains for further structural analysis.

Domain structure of gpNu1, a phage lambda DNA packaging protein.

The terminase enzyme from bacteriophage lambda is responsible for the insertion of a dsDNA genome into the confines of the viral capsid. The holoenzyme is composed of gpA and gpNu1 subunits in a gpA(1) x gpNu1(2) stoichiometry. While genetic studies have described regions within the two proteins responsible for DNA binding, capsid binding, and subunit interactions in the holoenzyme complex, biochemical characterization of these domains is limited. We have previously described the cloning, expression, and biochemical characterization of a soluble DNA binding domain of the terminase gpNu1 subunit (Met1 to Lys100) and suggested that the hydrophobic region spanning Lys100 to Pro141 defines a domain responsible for self-association interactions, and that is important for cooperative DNA binding [Yang et al. (1999) Biochemistry 38, 465-477]. We further suggested that the genetically defined gpA-interactive domain in the C-terminal half of the protein is limited to the C-terminal approximately 40 amino acids of gpNu1. Here we describe the cloning, expression, and biochemical characterization of gpNu1DeltaP141, a deletion mutant of gpNu1 that comprises the DNA binding domain and the putative hydrophobic self-assembly domain of the full-length protein. Purified gpNu1DeltaP141 shows a strong tendency to aggregate in solution; However, the protein remains soluble in 0.4 M guanidine hydrochloride, and circular dichroism (CD) and fluorescence spectroscopic studies demonstrate that the protein is folded under these conditions. Moreover, CD spectroscopy and thermally induced unfolding studies suggest that the DNA binding domain and the self-association domain represent independent folding domains of gpNu1DeltaP141. The mutant protein interacts weakly with the gpA subunit, but does not form a catalytically competent holoenzyme complex, suggesting that the C-terminal 40 residues are important for appropriate subunit interactions. Importantly, gpNu1DeltaP141 binds DNA tightly, but with less specificity than does full-length protein, and the data suggest that the C-terminal residues are further required for specific DNA binding activity. The implications of these results in the assembly of a functional holoenzyme complex are discussed.

Mutations in Nu1, the gene encoding the small subunit of bacteriophage lambda terminase, suppress the postcleavage DNA packaging defect of cosB mutations.

The linear double-stranded DNA molecules in lambda virions are generated by nicking of concatemeric intracellular DNA by terminase, the lambda DNA packaging enzyme. Staggered nicks are introduced at cosN to generate the cohesive ends of virion DNA. After nicking, the cohesive ends are separated by terminase; terminase bound to the left end of the DNA to be packaged then binds the empty protein shell, i.e., the prohead, and translocation of DNA into the prohead occurs. cosB, a site adjacent to cosN, is a terminase binding site. cosB facilitates the rate and fidelity of the cosN cleavage reaction by serving as an anchoring point for gpNu1, the small subunit of terminase. cosB is also crucial for the formation of a stable terminase-DNA complex, called complex I, formed after cosN cleavage. The role of complex I is to bind the prohead. Mutations in cosB affect both cosB functions, causing mild defects in cosN cleavage and severe packaging defects. The lethal cosB R3- R2- R1- mutation contains a transition mutation in each of the three gpNu1 binding sites of cosB. Pseudorevertants of lambda cosB R3- R2- R1- DNA contain suppressor mutations affecting gpNu1. Results of experiments that show that two such suppressors, Nu1ms1 and Nu1ms3, do not suppress the mild cosN cleavage defect caused by the cosB R3- R2- R1- mutation but strongly suppress the DNA packaging defect are presented. It is proposed that the suppressing terminases, unlike the wild-type enzyme, are able to assemble a stable complex I with cosB R3- R2- R1- DNA. Observations on the adenosine triphosphatase activities and protease susceptibilities of gpNu1 of the Nu1ms1 and Nu1ms3 terminases indicate that the conformation of gpNu1 is altered in the suppressing terminases.

Assembly of a nucleoprotein complex required for DNA packaging by bacteriophage lambda.

A critical step in the assembly of bacteriophage lambda is the excision of a single genome from a concatemeric DNA precursor and insertion of genomic DNA into an empty viral capsid. DNA packaging is mediated by the lambda proteins gpNu1 and gpA, which form an enzyme complex known as terminase. Initiation of the packaging process requires assembly of the terminase subunits onto cos, the lambda DNA packaging sequence, and nicking of the duplex, thus forming the 12-base-pair "sticky" ends of the mature genome. We have utilized gel-retardation techniques to examine the interaction of gpNu1, gpA, and terminase holoenzyme with DNA. Our data demonstrate that gpNu1 interacts specifically with cos-containing DNA, forming three gel-retarded complexes. Similarly, the larger gpA subunit binds to DNA, forming two complexes; however, this subunit forms similar complexes with DNA substrates of random sequence. All of the nucleoprotein complexes examined are disrupted by elevated concentrations of NaCl and we suggest that altered DNA binding is responsible for the extreme salt sensitivity of the endonuclease activity of the enzyme [Tomka, M. A., & Catalano, C. E. (1993) J. Biol. Chem. 268, 3056-3065]. DNA binding by each subunit is strongly affected by the presence of the other, with 10- and 3-fold increases in the affinity of gpNu1 and gpA, respectively, for DNA. Moreover, our data suggest that the terminase subunits interact in solution prior to DNA binding. Finally, we provide evidence that complex I, the first stable intermediate in the packaging pathway, is composed of the mature left genome end bound to the terminase subunits and demonstrate that dissociation of the complex is quite slow (t1/2 > 8 h). The significance of these data with respect to terminase-mediated genome packaging is discussed.

Isolation and characterization of the hyperthermostable serine protease, pyrolysin, and its gene from the hyperthermophilic archaeon Pyrococcus furiosus.

The hyperthermostable serine protease pyrolysin from the hyperthermophilic archaeon Pyrococcus furiosus was purified from membrane fractions. Two proteolytically active fractions were obtained, designated high (HMW) and low (LMW) molecular weight pyrolysin, that showed immunological cross-reaction and identical NH2-terminal sequences in which the third residue could be glycosylated. The HMW pyrolysin showed a subunit mass of 150 kDa after acid denaturation. Incubation of HMW pyrolysin at 95 degrees C resulted in the formation of LMW pyrolysin, probably as a consequence of COOH-terminal autoproteolysis. The 4194-base pair pls gene encoding pyrolysin was isolated and characterized, and its transcription initiation site was identified. The deduced pyrolysin sequence indicated a prepro-enzyme organization, with a 1249-residue mature protein composed of an NH2-terminal catalytic domain with considerable homology to subtilisin-like serine proteases and a COOH-terminal domain that contained most of the 32 possible N-glycosylation sites. The archaeal pyrolysin showed highest homology with eucaryal tripeptidyl peptidases II on the amino acid level but a different cleavage specificity as shown by its endopeptidase activity toward caseins, casein fragments including alphaS1-casein and synthetic peptides.

The lambda terminase enzyme measures the point of its endonucleolytic attack 47 +/- 2 bp away from its site of specific DNA binding, the R site.

lambda terminase is an ATP-interactive, site-specific endonuclease comprising the products of lambda genes Nu1 and A. Terminase binds to cos, at the junction of two chromosomes in a concatemer, catalyzes cos cleavage and initiates the packaging of lambda DNA into proheads. cos consists of a nicking domain, cosN, where terminase cleaves to regenerate the 12 nucleotide cohesive ends of mature lambda chromosomes and a binding domain, cosB, where terminase binds to 16 bp repeat sequences called R3, R2 and R1. Evidence is presented that terminase is a single-strand endonuclease that can nick DNA by one of two mechanisms, both of which require ATP. (i) When bound to any R site, terminase nicks the strand which, within that R site, is purine-rich; the position of this nick is 47 +/- 2 nucleotides away from the mid-point of that R site, measured in the 3' direction; (ii) enzymes that are not bound to R sites nick DNA within certain specific sequences that resemble cosN half sites. These two modes of action are nicely combined for the R3-bound protomer that nicks the bottom strand at position N1 in cosN since the interval between N1 and the R3 midpoint is 47 nucleotides. Within cosN, the bottom and top strand nicks are generated by a rigid protein couple with a 2-fold rotational symmetry. The location of both of these nicks, however, is gauged asymmetrically from R3, 47 nucleotides away. Again, R1 and R2 are separated by 47 bp and orient bound protomers towards each other but, unless the DNA between these R sites is lengthened, the enzymes do not nick, indicating an inhibitory gpA-gpNu1 apposition.

A new procedure for the purification of the bacteriophage lambda terminase enzyme and its subunits. Properties of gene product A, the large subunit.

New methods for the purification of highly active bacteriophage lambda terminase holoenzyme, and its individual subunits, gene products (gp) A and gpNu1, have been developed. These methods are rapid, simple, reproducible, and give high yields of unaggregated protein from small volumes of culture. The procedures involve fractionation of extracts of Escherichia coli strains harboring plasmids engineered to overproduce the respective proteins. All purified proteins exist as monomers or dimers at moderate concentrations. At concentrations where holoenzyme efficiently promotes in vitro cosN-cleavage and lambda DNA packaging, gpA displays neither of these activities unless supplemented with gpNu1 and the E. coli protein integration host factor. At high protein concentrations, however, gpA can promote cos-cleavage by itself. Although gpNu1 itself cannot promote either cosN-cleavage or DNA packaging, it does modulate these activities of gpA. GpA is a DNA-stimulated ATPase whose catalytic parameters closely resemble those of the holoenzyme. Like the holoenzyme, gpA displays a DNA helicase activity which is able to melt the annealed cosN overhangs. Certain preparations of gpA appear to undergo a time-dependent amino-terminal clipping at discrete sites even in the presence of as many as four protease inhibitors and at low temperature.

Isolation and characterization of mutations in the bacteriophage lambda terminase genes

The terminase enzyme of bacteriophage lambda is a hetero-oligomeric protein which catalyzes the site-specific endonucleolytic cleavage of lambda DNA and its packaging into phage proheads; it is composed of the products of the lambda Nul and A genes. We have developed a simple method to select mutations in the terminase genes carried on a high-copy-number plasmid, based on the ability of wild-type terminase to kill recA strains of Escherichia coli. Sixty-three different spontaneous mutations and 13 linker insertion mutations were isolated by this method and analyzed. Extracts of cells transformed by mutant plasmids displayed variable degrees of reduction in the activity of one or both terminase subunits as assayed by in vitro lambda DNA packaging. A method of genetically mapping plasmid-borne mutations in the A gene by measuring their ability to rescue various lambda Aam phages showed that the A mutations were fairly evenly distributed across the gene. Mutant A genes were also subcloned into overproducing plasmid constructs, and it was determined that more than half of them directed the synthesis of normal amounts of full-length A protein. Three of the A gene mutants displayed dramatically reduced in vitro packaging activity only when immature (uncut) lambda DNA was used as the substrate; therefore, these mutations may lie in the endonuclease domain of terminase. Interestingly, the putative endonuclease mutations mapped in two distinct locations in the A gene separated by a least 400 bp.

Direct and general selection for lysogens of Escherichia coli by phage lambda recombinant clones.

We report a simple in vivo technique for introducing an antibiotic resistance marker into phage lambda. This technique could be used for direct selection of lysogens harboring recombinant phages from the Kohara lambda bank (a collection of ordered lambda clones carrying Escherichia coli DNA segments). The two-step method uses homologous recombination and lambda DNA packaging to replace the nonessential lambda DNA lying between the lysis genes and the right cohesive (cos) end with the neomycin phosphotransferase (npt) gene from Tn903. This occurs during lytic growth of the phage on a plasmid-containing host strain. Neomycin-resistant (npt+) recombinant phages are then selected from the lysates containing the progeny phage by transduction of a polA1 lambda lysogenic host strain to neomycin resistance. We have tested this method with two different Kohara lambda phage clones; in both cases, neomycin resistance cotransduced with the auxotrophic marker carried by the lambda clone, indicating complete genetic linkage. Linkage was verified by restriction mapping of purified DNA from a recombinant phage clone. We also demonstrate that insertion of the npt+ recombinant phages into the lambda prophage can be readily distinguished from insertion into bacterial chromosomal sequences.

HU and integration host factor function as auxiliary proteins in cleavage of phage lambda cohesive ends by terminase

HU and integration host factor (IHF) are small, basic heterodimeric DNA-binding proteins which participate in transcription initiation, DNA replication, and recombination. We constructed isogenic Escherichia coli strains in which HU, IHF, or both proteins were absent. Bacteriophage lambda did not grow in hosts lacking both HU and IHF. Phage DNA replication and late gene transcription were normal in the double mutants, but packaging of lambda DNA was defective. Mature phage DNA molecules were absent, indicating that terminase was unable to linearize lambda DNA. Phage variants carrying a small substitution near cos or the ohm1 mutation in the terminase gene, Nul, formed plaques on HU- IHF- strains. We propose that HU or IHF is required to establish the higher-order DNA-protein structure at cos that is the substrate for lambda terminase.

In vitro transposition of transposon Tn3.

Transposition of the ampicillin-resistant transposon Tn3 was reproduced in vitro using the Escherichia coli cell extract. In this cell-free system, we used plasmid DNA carrying mini-Tn3 as donor and phage lambda DNA as target and assayed for ampicillin-resistance transducing phages formed by cointegration of these DNA molecules. Ampicillin-resistance transducing phages, which were obtained by in vitro packaging of lambda DNA after the in vitro transposition reaction, were formed only in the presence of Tn3 transposase. The reaction required mini-Tn3 with the proper sequence and orientation of the terminal inverted repeats of Tn3. The reaction also required DNA synthesis but not RNA synthesis by E. coli RNA polymerase.

Lambda vectors for stable cloned gene expression.

The bacteriophage lambda offers a unique opportunity concurrently to minimize segregational instability in recombinant systems by chromosomal integration of the cloned gene and to achieve high cloned gene expression during an abortive lytic phase. Lysis leads approximately to a 100-fold amplification of the cloned gene. Cell lysis in the lytic state is blocked by a specific mutation (Sam), allowing the cell to maintain its integrity, and lambda DNA packaging is blocked by other mutations (Wam, Eam) that keep cloned genes open to transcription. In the presence of these mutations, extremely high levels of cloned beta-galactosidase (more than 15% of total cell protein) have been obtained during abortive lysis from vectors found to be essentially 100% stable for over 75 generations in the lysogenic phase.

Formation of λ Transducing Phage In Vitro: Involvement of DNA Gyrase1

We found that transducing phages carrying the gal or bio regions of the Escherichia coli genome were formed during in vitro packaging of endogenous lambda DNA. Structural analysis of the transducing phage genomes indicated that they were formed by abnormal excision of lambda prophage. Formation of transducing phages was stimulated by oxolinic acid, an inhibitor of DNA gyrase, implying that DNA gyrase participates in the abnormal excision of lambda prophage. When pBR322 DNA was added to the reaction mixture, transducing phages into which pBR322 had been inserted were produced at a high frequency. This reaction was also stimulated by oxolinic acid. Sequence analyses revealed that pBR322 is inserted into the sites of abnormal excision of the prophage. These results show that transducing phages can be formed by DNA gyrase-dependent illegitimate recombination in an in vitro system and that secondary recombination takes place frequently at the site where the first recombination occurs.

The interaction ofE.coliintegration host factor and λcosDNA: multiple complex formation and protein-induced bending

The interaction of E. coli's integration Host Factor (IHF) with fragments of lambda DNA containing the cos site has been studied by gel-mobility retardation and electron microscopy. The cos fragment used in the mobility assays is 398 bp and spans a region from 48,298 to 194 on the lambda chromosome. Several different complexes of IHF with this fragment can be distinguished by their differential mobility on polyacrylamide gels. Relative band intensities indicate that the formation of a complex between IHF and this DNA fragment has an equilibrium binding constant of the same magnitude as DNA fragments containing lambda's attP site. Gel-mobility retardation and electron microscopy have been employed to show that IHF sharply bends DNA near cos and to map the bending site. The protein-induced bend is near an intrinsic bend due to DNA sequence. The position of the bend suggests that IHF's role in lambda DNA packaging may be the enhancement of terminase binding/cos cutting by manipulating DNA structure.

The Nu1 subunit of bacteriophage lambda terminase.

The maturation and packaging of bacteriophage lambda DNA are catalyzed by the phage terminase enzyme. Terminase is composed of two protein subunits, gpNu1 and gpA. The holoenzyme is multifunctional in vitro; it binds to and cleaves lambda DNA at the cos site (where cos represents cohesive-end site), packages DNA into lambda proheads, and is also a DNA-dependent ATPase. The genes of the two subunits have been cloned separately into powerful expression vectors which allow for very high levels of protein overproduction. The gpNu1 protein has been purified to homogeneity and has a monomeric molecular weight of 21,200, in close agreement with the Mr of 20,444 expected from its amino acid sequence. Both gel filtration and sedimentation velocity centrifugation indicate that the native gpNu1 protein exists as a Mr greater than 500,000 aggregate. The sequence of the first 20 amino acids and the overall composition both match those predicted by the nucleotide sequence of the Nu1 gene. Purified gpNu1 is able to complement gpA-containing extracts in both lambda DNA packaging and cos cleavage assays. The Nu1 gene amino acid sequence predicts DNA binding by the protein, and gpNu1 does show specific binding to lambda DNA by filter binding assays. Also, as predicted from its sequence, gpNu1 exhibits ATPase activity; but in contrast to the holoenzyme, this activity is DNA-independent.

Bacteriophage lambda DNA packaging: a mutant terminase that is independent of integration host factor.

Lambda+ is able to grow in Escherichia coli cells lacking integration host factor (IHF), producing a burst of approximately 25% that produced in IHF+ cells. In vitro, however, we find that the lambda DNA packaging enzyme terminase is strongly dependent on IHF in both cos cleavage reactions and DNA packaging reactions. The cos59 mutation renders lambda dependent on IHF in vivo. The cos59 mutation is a deletion of 3 base pairs at the XmnI site in the cohesive end site (cos) of lambda. Variants of lambda cos59 that were able to grow in the absence of IHF were isolated and found to carry a mutation, called ms1, in the Nu1 gene, which codes for the small subunit of terminase. The Nu1ms1 mutation results in a change of the 40th amino acid of the Nu1 gene product from leucine to phenylalanine. The Nu1ms1 terminase was independent of IHF in packaging reactions in vitro. The results indicate that the mutation either renders terminase: (1) able to utilize some host protein other than IHF, or (2) totally independent of host factors.

The bacteriophage lambda cohesive end site: isolation of spacing/substitution mutations that result in dependence on Escherichia coli integration host factor.

Substitution, insertion and deletion mutations have been constructed at the XmnI restriction site in cos lambda. The XmnI site is located between cosB, the site where terminase binds lambda DNA; and cosN, the site where terminase introduces staggered nicks to generate cohesive ends. Substitution mutations and deletion of a base pair (a -1 change) do not obviously affect lambda growth and DNA packaging. Changes of -2, +2 and -3 render lambda unable to grow on host cells lacking integration host factor (IHF). The -3 mutant has a reduced burst size in IHF+ cells, due to a defect in the initiation of packaging. A -7 deletion mutation is lethal. Models for the basis of these mutational effects are discussed.

Bacteriophage lambda DNA packaging: The product of the FI gene promotes the incorporation of the prohead to the DNA-terminase complex

Lambda DNA packaging in vitro can be examined in stages. In a first step, λ DNA interacts with terminase to form a DNA-enzyme complex, called complex I. Upon addition of proheads, in a second step, a ternary complex, complex II, containing DNA, terminase and the prohead is formed. Finally, upon addition of the rest of the morphogenetic components, complete phages are assembled. We have investigated the effect of the FI gene product (gpFI) in these reactions and found that a stimulation in phage yield is observed when gpFI is included early in the reaction, at the time when DNA, terminase and proheads interact to form complex II. Measurements of complex II formation revealed that gpFI stimulated the rate of formation of this intermediate. gpFI was further shown to stimulate the addition of proheads to preformed complexes I to give complex II, but the protein did not stimulate complex I formation.

The Nul subunit of bacteriophage lambda terminase binds to specific sites in cos DNA.

The maturation and packaging of bacteriophage lambda DNA are under the control of the multifunctional viral terminase enzyme, which is composed of the protein products of Nu1 and A, the two most leftward genes of the phage chromosome. Terminase binds selectively to the cohesive end site (cos) of multimeric replicating lambda DNA and introduces staggered nicks to regenerate the 12-base single-stranded cohesive ends of the mature phage genome. The purified gpNu1 subunit of terminase forms specific complexes with cos lambda DNA. DNase I footprinting experiments showed that gpNu1 bound to three distinct regions near the extreme left end of the lambda chromosome. These regions coincided with two 16-base-pair sequences (CTGTCGTTTCCTTTCT) that were in inverted orientation, as well as a truncated version of this sequence. Bear et al. (J. Virol. 52:966-972,1984) isolated a mutant phage which contained a CG to TA transition at the 10th position of the rightmost 16-base-pair sequence, and this phage (termed lambda cos 154) exhibits a defect in DNA maturation when it replicates in Escherichia coli which is deficient in integration host factor. Footprinting experiments with cos 154 DNA showed that gpNu1 could not bind to the site which contained the mutation but could protect the other two sites. Since the DNA-packaging specificity of terminase resides in the gpNu1 subunit, these studies suggest that terminase uses these three sites as recognition sequences for specific binding to cos lambda.