Cementing proteins provide extra mechanical stabilization to viral cages.

The study of virus shell stability is key not only for gaining insights into viral biological cycles but also for using viral capsids in materials science. The strength of viral particles depends profoundly on their structural changes occurring during maturation, whose final step often requires the specific binding of 'decoration' proteins (such as gpD in bacteriophage lambda) to the viral shell. Here we characterize the mechanical stability of gpD-free and gpD-decorated bacteriophage lambda capsids. The incorporation of gpD into the lambda shell imparts a major mechanical reinforcement that resists punctual deformations. We further interrogate lambda particle stability with molecular fatigue experiments that resemble the sub-lethal Brownian collisions of virus shells with macromolecules in crowded environments. Decorated particles are especially robust against collisions of a few kBT (where kB is the Boltzmann's constant and T is the temperature ~300 K), which approximate those anticipated from molecular insults in the environment.

Single-particle visualization of assembly: I. Dimerization in a planar zone.

Summary Single-particle fluorescence microscopy of association/dissociation is required for analysis of biological assembly reactions. Toward achieving this goal, Wang et al. (J. Microsc., 2004, 213, 101-109) used molten agarose to concentrate thermally diffusing particles in a thin zone of solution next to the surface of a coverglass (plane of concentration). The present study details the first real-time, single-particle analysis of the association/dissociation of thermally diffusing particles in the plane of concentration. The test particles were procapsids of bacteriophage lambda (radius = 31 nm). Quantification of thermal motion was developed and used to determine whether co-diffusing particles were bound to each other. The data are explained by (1) the presence of a molten agarose-generated barrier that is 93-155 nm from the coverglass surface, and (2) non-random orientation of procapsid dimers in the plane of concentration.

The functional asymmetry of cosN, the nicking site for bacteriophage lambda DNA packaging, is dependent on the terminase binding site, cosB.

cosN is the site at which terminase, the DNA packaging enzyme of phage lambda, introduces staggered nicks into viral concatemeric DNA to initiate genome packaging. Although the nick positions and many of the base pairs of cosN show 2-fold rotational symmetry, cosN is functionally asymmetric. That is, the cosN G2C mutation in the left half-site (cosNL) causes a strong virus growth defect whereas the symmetrically disposed cosN C11G mutation in the right half-site (cosNR) does not affect virus growth. The experiments reported here test the proposal that the genetic asymmetry of cosN results from terminase interactions with cosB, a binding site to the right of cosN. In the presence of cosB, the left half-site mutation, cosN G2C, strongly affected the cos cleavage reaction, while the symmetric right half-site mutation, cosN C11G, had little effect. In the absence of cosB, the two mutations moderately reduced the rate of cos cleavage by the same amount. The results indicated that the functional asymmetry of cosNdepends on the presence of cosB. A model is discussed in which terminase-cosN interactions in the nicking complex are assisted by anchoring of terminase to cosB.

Biophysical characterization of the DNA binding domain of gpNu1, a viral DNA packaging protein.

Terminase enzymes are common to double-stranded DNA viruses. These enzymes "package" the viral genome into a pre-formed capsid. Terminase from bacteriophage lambda is composed of gpA (72.4 kDa) and gpNu1 (20.4 kDa) subunits. We have described the expression and biochemical characterization of gpNu1DeltaK100, a construct comprising the N-terminal 100 amino acids of gpNu1 (Yang, Q., de Beer, T., Woods, L., Meyer, J., Manning, M., Overduin, M., and Catalano, C. E. (1999) Biochemistry 38, 465-477). Here we present a biophysical characterization of this construct. Thermally induced loss of secondary and tertiary structures is fully reversible. Surprisingly, although loss of tertiary structure is cooperative, loss of secondary structure is non-cooperative. NMR and limited proteolysis data suggest that approximately 30 amino acids of gpNu1DeltaK100 are solvent-exposed and highly flexible. We therefore constructed gpNu1DeltaE68, a protein consisting of the N-terminal 68 residues of gpNu1. gpNu1DeltaE68 is a dimer with no evidence of dissociation or further aggregation. Thermally induced unfolding of gpNu1DeltaE68 is reversible, with concomitant loss of both secondary and tertiary structure. The melting temperature increases with increasing protein concentration, suggesting that dimerization and folding are, at least in part, coupled. The data suggest that gpNu1DeltaE68 represents the minimal DNA binding domain of gpNu1. We further suggest that the C-terminal approximately 30 residues in gpNu1DeltaK100 adopt a pseudo-stable alpha-helix that extends from the folded core of the protein. A model describing the role of this helix in the assembly of the packaging apparatus is discussed.

The terminase enzyme from bacteriophage lambda: a DNA-packaging machine.

This review focuses on the biochemical, biophysical, and catalytic properties of terminase, an enzyme involved in bacteriophage lambda genome packaging. The holoenzyme possesses ATPase, DNA strand-separation, and site-specific nuclease activities that work in concert to insert a viral genome into the confines of a performed capsid. Moreover, the terminase subunits are part of a series of nucleoprotein complexes involved in genome packaging, including remarkably stable intermediates that transition to a highly mobile DNA packaging 'machine.' Models for the assembly and interconversion of these complexes are presented. Interactions between the catalytic sites in the enzyme complex, and modulation of these catalytic activities as it relates to the assembly and relative stability of the packaging intermediates are discussed. This ordered progression of nucleoprotein intermediates is a common theme in biology as demonstrated by mechanistic similarities between viral DNA packaging, the initiation of chromosomal replication, and the initiation of transcription. Terminase is thus part of a growing number of examples of biological 'machines' or molecular 'motors.'

Kinetic characterization of the GTPase activity of phage lambda terminase: evidence for communication between the two "NTPase" catalytic sites of the enzyme.

The terminase enzyme from bacteriophage lambda is responsible for the insertion of viral DNA into the confined space within the capsid. The enzyme is composed of the virally encoded proteins gpA (73.3 kDa) and gpNu1 (20.4 kDa) isolated as a gpA(1).gpNu1(2) holoenzyme complex. Lambda terminase possesses a site-specific nuclease activity, an ATP-dependent DNA strand-separation activity, and an ATPase activity that must work in concert to effect genome packaging. We have previously characterized the ATPase activity of the holoenzyme and have identified catalytic active sites in each enzyme subunit [Tomka and Catalano (1993) Biochemistry 32, 11992-11997; Hwang et al. (1996) Biochemistry 35, 2796-2803]. We have noted that GTP stimulates the ATPase activity of the enzyme, and terminase-mediated GTP hydrolysis has been observed. The studies presented here describe a kinetic analysis of the GTPase activity of lambda terminase. GTP hydrolysis by the enzyme requires divalent metal, is optimal at alkaline pH, and is strongly inhibited by salt. Interestingly, while GTP can bind to the enzyme in the absence of DNA, GTP hydrolysis is strictly dependent on the presence of polynucleotide. Unlike ATP hydrolysis that occurs at both subunits of the holoenzyme, a single catalytic site is observed in the steady-state kinetic analysis of GTPase activity (k(cat) approximately 37 min(-)(1); K(m) approximately 500 microM). Moreover, while GTP stimulates ATP hydrolysis (apparent K(D) approximately 135 microM for GTP binding), all of the adenosine nucleotides examined strongly inhibit the GTPase activity of the enzyme. The data presented here suggest that the two "NTPase" catalytic sites in terminase holoenzyme communicate, and we propose a model describing allosteric interactions between the two sites. The biological significance of this interaction with respect to the assembly and disassembly of the multiple nucleoprotein packaging complexes required for virus assembly is discussed.

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.

Cloning, expression, and biochemical characterization of hexahistidine-tagged terminase proteins.

The terminase enzyme from bacteriophage lambda is composed of two viral proteins (gpA, 73.2 kDa; gpNu1, 20.4 kDa) and is responsible for packaging viral DNA into the confines of an empty procapsid. We are interested in the genetic, biochemical, and biophysical properties of DNA packaging in phage lambda and, in particular, the nucleoprotein complexes involved in these processes. These studies require the routine purification of large quantities of wild-type and mutant proteins in order to probe the molecular mechanism of DNA packaging. Toward this end, we have constructed a hexahistidine (hexa-His)-tagged terminase holoenzyme as well as hexa-His-tagged gpNu1 and gpA subunits. We present a simple, one-step purification scheme for the purification of large quantities of the holoenzyme and the individual subunits directly from the crude cell lysate. Importantly, we have developed a method to purify the highly insoluble gpNu1 subunit from inclusion bodies in a single step. Hexa-His terminase holoenzyme is functional in vivo and possesses steady-state and single-turnover ATPase activity that is indistinguishable from wild-type enzyme. The nuclease activity of the modified holoenzyme is near wild type, but the reaction exhibits a greater dependence on Escherichia coli integration host factor, a result that is mirrored in vivo. These results suggest that the hexa-His-tagged holoenzyme possesses a mild DNA-binding defect that is masked, at least in part, by integration host factor. The mild defect in hexa-His terminase holoenzyme is more significant in the isolated gpA-hexa-His subunit that does not appear to bind DNA. Moreover, whereas the hexa-His-tagged gpNu1 subunit may be reconstituted into a holoenzyme complex with wild-type catalytic activities, gpA-hexa-His is impaired in its interactions with the gpNu1 subunit of the enzyme. The results reported here underscore that a complete biochemical characterization of the effects of purification tags on enzyme function must be performed prior to their use in mechanistic studies.

Cloning, expression, and characterization of a DNA binding domain of gpNu1, a phage lambda DNA packaging protein.

Terminase is an enzyme from bacteriophage lambda that is required for insertion of the viral genome into an empty pro-capsid. This enzyme is composed of the viral proteins gpNu1 (20.4 kDa) and gpA (73.3 kDa) in a holoenzyme complex. Current models for terminase assembly onto DNA suggest that gpNu1 binds to three repeating elements within a region of the lambda genome known as cosB which, in turn, stimulates the assembly of a gpA dimer at the cosN subsite. This prenicking complex is the first of several stable nucleoprotein intermediates required for DNA packaging. We have noted a hydrophobic region within the primary amino acid sequence of the terminase gpNu1 subunit and hypothesized that this region constitutes a protein-protein interaction domain required for cooperative assembly at cosB and that is also responsible for the observed aggregation behavior of the isolated protein. We therefore constructed a mutant of gpNu1 in which this hydrophobic "domain" has been deleted in order to test these hypotheses. The deletion mutant protein, gpNu1DeltaK, is fully soluble and, unlike full-length protein, shows no tendency toward aggregation; However, the protein is a dimer under all experimental conditions examined as determined by gel permeation and sedimentation equilibrium analysis. The truncated protein is folded with evidence of secondary and tertiary structural elements by circular dichroism and NMR spectroscopy. While physical and biological assays demonstrate that gpNu1DeltaK does not interact with the terminase gpA subunit, the deletion mutant binds with specificity to cos-containing DNA. We have thus constructed a deletion mutant of the phage lambda terminase gpNu1 subunit which constitutes a highly soluble DNA binding domain of the protein. We further propose that the hydrophobic amino acids found between Lys100 and Pro141 define a self-association domain that is required for the assembly of stable nucleoprotein packaging complexes and that the C-terminal tail of the protein defines a distinct gpA-binding site that is responsible for terminase holoenzyme formation.

The phage lambda terminase enzyme: 1. Reconstitution of the holoenzyme from the individual subunits enhances the thermal stability of the small subunit.

The terminase enzyme from bacteriophage lambda is a hetero-trimeric complex composed of the viral gpA and gpNu1 proteins (gpA1.gpNu1(2)) and is responsible for packaging a single genome within the viral capsid. Current expression systems for these proteins require thermal induction which may be responsible for the formation of insoluble aggregates observed in E. coli. We report the re-cloning of the terminase subunits into vectors which allow low temperature induction. While this has resulted in increased solubility of the large gpA subunit of the enzyme, the small gpNu1 subunit remains insoluble under all conditions examined. This paper describes the solublization of gpNu1 with guanidinium hydrochloride and purification of the protein to homogeneity. Reconstitution of the enzyme from the individually purified subunits yields a catalytically-competent complex which exhibits activity identical to wild-type enzyme. Thermal denaturation of the proteins was monitored by circular dichroism (CD) spectroscopy and demonstrates that while unfolding of gpA is irreversible, the gpNu1 subunit refolds into a conformation which is essentially identical to the pre-heated protein. Moreover, while denaturation of gpA is highly cooperative, the small subunit unfolds over a wide temperature range and with thermodynamic parameters lower than expected for a small globular protein. Thermally-induced denaturation of the enzyme reconstituted from the individual subunits is highly cooperative with no evidence of multiple transitions. Our data demonstrate that the terminase subunits directly interact in solution, and that this interaction alters the thermal stability of the smaller gpNu1 subunit. The implication of these results with respect to assembly of a catalytically competent enzyme complex are discussed.

The phage lambda terminase enzyme: 2. Refolding of the gpNu1 subunit from the detergent-denatured and guanidinium hydrochloride-denatured state yields different oligomerization states and altered protein stabilities.

The terminase enzyme from bacteriophage lambda is responsible for packaging a single genome within the viral capsid. Gold and co-workers have developed a scheme for the solubilization of the small terminase subunit (gpNu1) from inclusion bodies using the strong detergent sarkosyl and purification of the protein to homogeneity (gpNu1SRK) (Parris et al., J Biol Chem 1994;269:13564-13574). We have developed a similar purification scheme except that guanidinium hydrochloride was used to denature the insoluble protein (gpNu1GDN). The circular dichroism (CD) spectra of both protein preparations suggest that they are predominantly alpha-helical when purified and stored in Tris buffers. Moreover, thermal denaturation of the proteins thus purified yielded similar thermodynamic parameters for unfolding (T(m), delta Hm and delta Sm of unfolding of approximately 306 K, approximately 22 kcal/mol and approximately 70 cal/mol.K, respectively). Interestingly, however, when the proteins were purified and stored in imidazole buffers, the gpNu1SRK preparation lost a significant amount of secondary structure and was more stable to both thermally-induced and guanidinium HCl-induced denaturation than was gpNu1GDN. The purified gpNu1 monomers oligomerize into apparent tetramers and hexamers in solution and the distribution between these two oligomeric states and into higher order aggregates depends upon buffer composition, salt concentration and protein concentration. Moreover, differences in the oligomerization state of gpNu1SRK and gpNu1GDN under identical buffer conditions were observed. The significance of these results with respect to the biological role of the phage lambda gpNu1 protein are discussed.

Kinetic characterization of the strand separation ("helicase") activity of the DNA packaging enzyme from bacteriophage lambda.

Bacteriophage lambda is assembled from preformed viral capsids (proheads), tails, and genomes that are excised from a concatemeric DNA precursor. The enzyme responsible for insertion of the genome into the precapsid is known as terminase. This enzyme possesses site-specific endonuclease, ATPase, and DNA strand separation ("helicase") catalytic activities, which work in concert to excise and package a single viral genome during phage assembly. We have previously characterized the endonuclease [Tomka, M. A., & Catalano, C. E. (1993) J. Biol. Chem. 268, 3056-3065] and ATPase [Tomka, M. A., & Catalano, C. E. (1993) Biochemistry 32, 11992-11997] catalytic activities of lambda terminase and present here similar studies on the strand separation activity of the enzyme. Strand separation requires terminase, divalent metal, and adenosine nucleotides with a hydrolyzable beta,gamma-phosphate bond. Two apparent binding sites for ATP-mediated strand separation were identified, one of which appears to be distinct from the high- and low-affinity sites previously observed for ATP hydrolysis [Hwang, Y., Catalano, C. E., & Feiss, M. (1995) Biochemistry 35, 2796-2803]. Salt stimulates the reaction at low concentrations but is strongly inhibitory at elevated concentrations, presumably due to impaired DNA binding. The above results are identical with either a complex DNA mixture (a nicked, annealed DNA duplex in the presence of excess nonspecific DNA) or a purified DNA substrate; however, a kinetic analysis of the reaction revealed that the observed rate was approximately 5-fold greater with the purified DNA substrate. Moreover, while Escherichia coli integration host factor (IHF) stimulates terminase-mediated strand separation with both substrates, the observed stimulation is more pronounced with the complex DNA mixture (10-fold rate increase) than the purified DNA substrate (5-fold rate increase). Our data are consistent with a model where IHF binding to the terminase assembly site forms a binary protein.DNA complex readily distinguishable from bulk DNA. The implications of these results to the process of DNA packaging in bacteriophage lambda are discussed.

Kinetic analysis of the endonuclease activity of phage lambda terminase: assembly of a catalytically competent nicking complex is rate-limiting.

The terminase enzyme from bacteriophage lambda is responsible for excision of a single genome from a concatameric DNA precursor and its insertion into an empty viral procapsid. The enzyme possesses a site-specific endonuclease activity which is responsible for excision of the viral genome and the formation of the 12 base-pair single-stranded "sticky" ends of mature lambda DNA. We have previously reported a kinetic analysis of the endonuclease activity of lambda terminase which showed an enzyme concentration-dependent change in the kinetic time course of the reaction [Tomka, M. A., & Catalano, C. E. (1993b) J. Biol. Chem. 268, 3056-3065]. We presented a model which suggested that the rate-limiting step in the nuclease reaction was the assembly of a catalytically competent prenicking complex. Here, we provide additional evidence for a slow assembly step in the nuclease reaction and demonstrate that the observed rate is affected by protein concentration, but not by the length of the DNA substrate. Consistent with our model, preincubation of terminase with DNA also yields an observable fast phase of the reaction, but only when large (> or = 3 kb) DNA substrates are used. Finally, we present data which demonstrate that phage lambda terminase can efficiently utilize DNA from the closely related phage phi21 as an endonuclease substrate and that the enzyme binds efficiently to the cosB region of both phage genomes. The implications of these results with respect to the assembly of a catalytically competent nucleoprotein complex required to initiate genome packaging are discussed.

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.

Kinetic and mutational dissection of the two ATPase activities of terminase, the DNA packaging enzyme of bacteriophage Chi.

Terminase the DNA packaging enzyme of bacteriophage chi, is a heteromultimer of gpNul (21 kDa) and gpA (74 kDa) subunits, encoded by the chi Nul and A genes, respectively. Sequence comparisons indicate that both gpNu1 and gpA have a match to the P-loop motif of ATPase centers, which is a glycine-rich segment followed by a lysine. By site-specific mutagenesis, we changed the lysines of the putative P-loops of gpNul (k35) and gpA (K497) to arginine, alanine, or aspartic acid, and studied the mutant enzymes by kinetic analysis and photochemical cross-linking with 8-azido-ATP. Both the gpNul and gpA subunits of wild-type terminase were covalently modified with 8-N3[32P] ATP in the presence of UV light. Saturation occurred with apparent dissociation constants of 508 and 3.5 microM for gpNul and gpA, resepctively. ATPase assays showed two activities: a low-affinity activity (Km=469 microM), and a high-affinity activity (Km=4.6 microM). The gpNul K35A and gpNul K35D mutant terminases showed decreased activity in the low-affinity ATPase activity. The reduced activities of these enzymes were recovered when 10 times more DNA was added, suggesting that the primary defect of the enzymes is alteration of the nonspecific, double-stranded DNA binding activity of terminase. ATPase assays and photolabeling of the gpA K497A and gpA K497D mutant terminases showed reduced affinity for ATP at the high-affinity site which was not restored by increased DNA. In summary, the results indicate the presence of a low-affinity, DNA-stimulated ATPase center in gpNul, and a high-affinity site in gpA.

Inactivation of DNA polymerase alpha-primase by acrolein: loss of activity depends on the DNA substrate.

We have utilized acrolein as a model compound to examine the biochemical behavior of chemically-modified DNA polymerase alpha-primase complex (pol alpha). We have found that acrolein irreversibly inactivates the DNA synthetic capacity of pol alpha polymerase in a time- and concentration-dependent manner. Double-stranded DNA protects pol alpha polymerase from inactivation when present during acrolein exposure, but single-stranded DNA, dATP and ATP do not. Strikingly, the activity of pol alpha polymerase is strongly dependent upon the DNA substrate utilized to assay catalytic activity after exposure to the aldehyde. The primase activity of pol alpha is also inactivated by exposure to acrolein, but the observed rate of inactivation is slower than that seen for DNA synthesis. Competitive labeling studies with [14C] iodoacetamide suggest that acrolein inactivation of the enzyme is mediated through the modification of protein sulfhydryl groups.

Role of gpFI protein in DNA packaging by bacteriophage lambda.

One of the final steps in the assembly of bacteriophage lambda is the excision of a single genome from a concatemeric DNA precursor and insertion of this monomer into a preformed capsid. Terminase enzymes are common to all of the double-stranded DNA phages, and in lambda this enzyme is responsible for both excision of a genome monomer from the concatemer and its insertion into the pro-capsid. We have previously demonstrated that the endonuclease activity of lambda terminase (cos-cleavage) was stoichiometric with enzyme and postulated that this was due to formation of a stable, postcleavage enzyme.DNA intermediate (complex I) (Tomka & Catalano, 1993b). Bacteriophage lambda gpFI protein is required for efficient assembly of the virus, and current models suggest that this protein increases the rate of pro-capsid binding to complex I. We show here that gpFI markedly stimulates cos-cleavage by lambda terminase, even in the absence of viral pro-capsids. Importantly, the observed increase in nicking activity did not result from an increase in the rate of cos-cleavage, but rather by an increase in turnover by the enzyme. These data suggest that gpFI destabilizes complex I, thus allowing terminase release from cos and catalytic turnover by the enzyme. The implications of these results with respect to terminase assembly onto viral DNA, nicking of the duplex, and subsequent translocation during packaging are discussed.

Virus DNA packaging: the strategy used by phage lambda.

Phage lambda, like a number of other large DNA bacteriophages 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 co-ordinated with DNA packaging, the process of translocation of the cut DNA into the preformed capsid precursor, the prohead. A key player in the lambda DNA packaging process is the phage-encoded enzyme terminase, which is involved in (i) recognition of the concatemeric lambda 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 to 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 endonuclease 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, gpNu1, 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 lambda has aspects that differ from other lambda DNA transactions. Unlike the site-specific recombination system of lambda, for DNA packaging the initial site-specific protein assemblage gives way to a mobile, translocating complex, and unlike the DNA replication system of lambda, the same protein machinery is used for both initiation and translocation during lambda DNA packaging.

Kinetic characterization of the ATPase activity of the DNA packaging enzyme from bacteriophage lambda.

Terminases are enzymes common to all of the complex double-stranded DNA viruses and are required for viral assembly. These enzymes function to excise a single viral genome from a concatemeric DNA precursor and package it into a preformed protective protein shell or capsid. ATP hydrolysis by these enzymes has been described and appears to be critical to the packaging process. We have previously characterized the endonuclease activity of purified terminase from bacteriophage lambda [Tomka, M. A., & Catalano, C. E. (1993) J. Biol. Chem. 268, 3056-3065], and we describe here a kinetic characterization of the ATPase activity of the enzyme. lambda Terminase possesses a DNA-stimulated ATPase activity and hydrolyzes ATP to ADP and Pi. This activity requires divalent metal and is supported by all of the group IIa metals examined, as well as Mn2+. The reaction is also stimulated by NaCl, GTP, and dGTP. Of note is that neither of the guanosine nucleotides is hydrolyzed by the enzyme, while dATP is hydrolyzed at a rate comparable to that of ATP. Kinetic analysis of the ATPase activity revealed two apparent binding sites for ATP hydrolysis. The high-affinity site (Km = 5 microM) and low-affinity site (Km approximately 1.3 mM) hydrolyze ATP with kcat = 3 and 16 min-1, respectively. While the high-affinity site is unaffected by the presence of DNA, ATP hydrolysis at the low-affinity site is stimulated by DNA, which results from both a decrease in the Km and a concomitant increase in the kcat of the reaction.(ABSTRACT TRUNCATED AT 250 WORDS)