Terminase Enzyme Research Articles

Biophysical and structural characterization of a multifunctional viral genome packaging motor.

The large dsDNA viruses replicate their DNA as concatemers consisting of multiple covalently linked genomes. Genome packaging is catalyzed by a terminase enzyme that excises individual genomes from concatemers and packages them into preassembled procapsids. These disparate tasks are catalyzed by terminase alternating between two distinct states-a stable nuclease that excises individual genomes and a dynamic motor that translocates DNA into the procapsid. It was proposed that bacteriophage λ terminase assembles as an anti-parallel dimer-of-dimers nuclease complex at the packaging initiation site. In contrast, all characterized packaging motors are composed of five terminase subunits bound to the procapsid in a parallel orientation. Here, we describe biophysical and structural characterization of the λ holoenzyme complex assembled in solution. Analytical ultracentrifugation, small angle X-ray scattering, and native mass spectrometry indicate that 5 subunits assemble a cone-shaped terminase complex. Classification of cryoEM images reveals starfish-like rings with skewed pentameric symmetry and one special subunit. We propose a model wherein nuclease domains of two subunits alternate between a dimeric head-to-head arrangement for genome maturation and a fully parallel arrangement during genome packaging. Given that genome packaging is strongly conserved in both prokaryotic and eukaryotic viruses, the results have broad biological implications.

Physical and Functional Characterization of a Viral Genome Maturation Complex

Genome packaging is strongly conserved in the complex double-stranded DNA viruses, including the herpesviruses and many bacteriophages. In these cases, viral DNA is packaged into a procapsid shell by a terminase enzyme. The packaging substrate is typically a concatemer composed of multiple genomes linked in a head-to-tail fashion, and terminase enzymes perform two essential functions: 1) excision of a unit length genome from the concatemer (genome maturation) and 2) translocation of the duplex into a procapsid (genome packaging). While the packaging motors have been described in some detail, the maturation complexes remain ill characterized. Here we describe the assembly, physical characteristics, and catalytic activity of the λ-genome maturation complex. The λ-terminase protomer is composed of one large catalytic subunit tightly associated with two DNA recognition subunits. The isolated protomer binds DNA weakly and does not discriminate between nonspecific DNA and duplexes that contain the packaging initiation sequence, cos. The Escherichia coli integration host factor protein (IHF) is required for efficient λ-development in vivo and a specific IHF recognition sequence is found within cos. We show that IHF and the terminase protomer cooperatively assemble at the cos site and that the small terminase subunit plays the dominant role in complex assembly. Analytical ultracentrifugation analysis reveals that the maturation complex is composed of four protomers and one IHF heterodimer bound at the cos site. Tetramer assembly activates the cos-cleavage nuclease activity of the enzyme, which matures the genome end in preparation for packaging. The stoichiometry and catalytic activity of the complex is reminiscent of the type IIE and IIF restriction endonucleases and the two systems may share mechanistic features. This study, to our knowledge, provides our first detailed glimpse into the structural and functional features of a viral genome maturation complex, an essential intermediate in the development of complex dsDNA viruses.

Molecular Dissection of the Forces Responsible for Viral Capsid Assembly and Stabilization by Decoration Proteins.

Complex double-stranded DNA viruses utilize a terminase enzyme to package their genomes into a preassembled procapsid shell. DNA packaging triggers a major conformational change in the proteins assembled into the shell and most often subsequent addition of a decoration protein that is required to stabilize the structure. In bacteriophage λ, DNA packaging drives a procapsid expansion transition to afford a larger but fragile shell. The gpD decoration protein adds to the expanded shell as trimeric spikes at each of the 140 three-fold axes. The spikes provide mechanical strength to the shell such that it can withstand the tremendous internal forces generated by the packaged DNA in addition to environmental insults. Hydrophobic, electrostatic, and aromatic-proline noncovalent interactions have been proposed to mediate gpD trimer spike assembly at the expanded shell surface. Here, we directly examine each of these interactions and demonstrate that hydrophobic interactions play the dominant role. In the course of this study, we unexpectedly found that Trp308 in the λ major capsid protein (gpE) plays a critical role in shell assembly. The gpE-W308A mutation affords a soluble, natively folded protein that does not further assemble into a procapsid shell, despite the fact that it retains binding interactions with the scaffolding protein, the shell assembly chaparone protein. The data support a model in which the λ procapsid shell assembles via cooperative interaction of monomeric capsid proteins, as observed in the herpesviruses and phages such as P22. The significance of the results with respect to capsid assembly, maturation, and stability is discussed.

Cytomegalovirus.

To consider new treatment options for cytomegalovirus (CMV) infection, review recent trials, and anticipate their use in clinical practice, focussing on bone marrow transplantation, congenital infection, and intervention during pregnancy. Three double-blind randomized placebo-controlled phase 2 proof-of-concept studies have each identified a novel antiviral drug with activity against CMV infection in bone marrow transplant patients. One of these (brincidofovir) inhibits the DNA polymerase that is the target of the currently licensed drug ganciclovir. Another new drug (maribavir) inhibits a protein kinase which, coincidentally, is the enzyme responsible for activating ganciclovir through phosphorylation. The third drug (letermovir) inhibits the terminase enzyme complex responsible for packaging unit length DNA into assembling virions.In addition, in a double-blind randomized placebo-controlled trial in neonates with symptomatic congenital CMV infection, a 6-month course of valganciclovir was superior to the standard 6-week course of the same drug. In pregnant women with primary CMV infection, administration of hyperimmune immunoglobulin did not significantly reduce transmission of CMV across the placenta. The ability to diagnose CMV infections reliably in different clinical settings through application of molecular laboratory methods has ushered in new ways of evaluating potential new treatments for CMV. Several of these may help control the diseases caused by this important human pathogen.

Structural and Biochemical Characterization of Phage λ FI Protein (gpFI) Reveals a Novel Mechanism of DNA Packaging Chaperone Activity

One of the final steps in the morphogenetic pathway of phage λ is the packaging of a single genome into a preformed empty head structure. In addition to the terminase enzyme, the packaging chaperone, FI protein (gpFI), is required for efficient DNA packaging. In this study, we demonstrate an interaction between gpFI and the major head protein, gpE. Amino acid substitutions in gpFI that reduced the strength of this interaction also decreased the biological activity of gpFI, implying that this head binding activity is essential for the function of gpFI. We also show that gpFI is a two-domain protein, and the C-terminal domain is responsible for the head binding activity. Using nuclear magnetic resonance spectroscopy, we determined the three-dimensional structure of the C-terminal domain and characterized the helical nature of the N-terminal domain. Through structural comparisons, we were able to identify two previously unannotated prophage-encoded proteins with tertiary structures similar to gpFI, although they lack significant pairwise sequence identity. Sequence analysis of these diverse homologues led us to identify related proteins in a variety of myo- and siphophages, revealing that gpFI function has a more highly conserved role in phage morphogenesis than was previously appreciated. Finally, we present a novel model for the mechanism of gpFI chaperone activity in the DNA packaging reaction of phage λ.

Assembly of a Genome Packaging Motor on Viral DNA Influences Packaging Mechanism

During virus assembly, a single genome is packaged into a preformed capsid by a terminase enzyme. Presently, the mechanistic details of the packaging motor are unknown. Single molecule experiments have demonstrated that viral DNA packaging motors are extremely powerful, capable of packaging dsDNA to over 20 atmospheres. Therefore, the mechanistic details of DNA packaging are of interest from both a biological (drug target), and engineering (nanomachine) standpoint. Bacteriophage Lambda (λ) is a model system for the study of dsDNA viruses, including herpesvirus and many bacteriophage. λ terminase is a multifunction enzyme complex with catalytic activities required to (1) site-specifically bind the viral genome, (2) carry out nuclease and helicase activities on the viral genome, (3) bind the empty procapsid, and (4) hydrolyze ATP and translocate viral DNA into the procapsid. The holoenzyme consists of proteins gpA and gpNu1 in a 1:2 ratio called the heterotrimeric protomer. The functional motor is composed of four protomers that assemble into a ring at the packaging initiation site of viral DNA.Here we describe physio-kinetic studies on terminase protomer assembly into a catalytically-competent genome maturation and packaging complex. Assembly of the protomer on viral DNA is much faster than previously thought and affords a motor that packages DNA with high specificity. Furthermore, the packaging ATPase activity of the protomer is down-regulated until both the viral genome and empty procapsid are mated by terminase in preparation for packaging. The ring-tetramer can be artificially assembled in vitro in the absence of DNA and is functional to package DNA. We find that there are important differences between ring and protomer with assembly of a motor on DNA influencing the packaging mechanism. Our results suggest that the protomeric unit is the biologically relevant species in vivo.

A Mutation in U L 15 of Herpes Simplex Virus 1 That Reduces Packaging of Cleaved Genomes

Herpesvirus genomic DNA is cleaved from concatemers that accumulate in infected cell nuclei. Genomic DNA is inserted into preassembled capsids through a unique portal vertex. Extensive analyses of viral mutants have indicated that intact capsids, the portal vertex, and all components of a tripartite terminase enzyme are required to both cleave and package viral DNA, suggesting that DNA cleavage and packaging are inextricably linked. Because the processes have not been functionally separable, it has been difficult to parse the roles of individual proteins in the DNA cleavage/packaging reaction. In the present study, a virus bearing the deletion of codons 400 to 420 of U(L)15, encoding a terminase component, was analyzed. This virus, designated vJB27, failed to replicate on noncomplementing cells but cleaved concatemeric DNA to ca. 35 to 98% of wild-type levels. No DNA cleavage was detected in cells infected with a U(L)15-null virus or a virus lacking U(L)15 codons 383 to 385, comprising a motif proposed to couple ATP hydrolysis to DNA translocation. The amount of vJB27 DNA protected from DNase I digestion was reduced compared to the wild-type virus by 6.5- to 200-fold, depending on the DNA fragment analyzed, thus indicating a profound defect in DNA packaging. Capsids containing viral DNA were not detected in vJB27-infected cells, as determined by electron microscopy. These data suggest that pU(L)15 plays an essential role in DNA translocation into the capsid and indicate that this function is separable from its role in DNA cleavage.

Dissecting Lambda Terminase: A Viral DNA Packaging Motor

During virus assembly, a single genome is packaged into a preformed capsid by a terminase enzyme. Presently, the mechanistic details of the packaging motor are unknown. Single molecule experiments have demonstrated that viral DNA packaging motors are among the most powerful currently known, capable of packaging dsDNA to over 20 atmospheres within the capsid. Therefore, the mechanistic details of DNA packaging are of interest from both a biological (drug target), and engineering (nanomachine) standpoint. Bacteriophage Lambda (λ) is a model system for the study of dsDNA viruses, including herpesvirus and many bacteriophage. λ terminase is a multifunction enzyme complex with catalytic activities required to (1) recognize the viral genome, (2) prepare the viral genome for packaging, (3) recognize the empty procapsid, and (4) translocate viral DNA into the empty procapsid. The enzyme consists of two proteins, gpA and gpNu1 in a 1:2 ratio known as the heterotrimeric protomer; the motor functions as a tetramer of protomers assembled into a ring complex.Here, we study the λ terminase in vitro and characterize macromolecular assembly, genome packaging, ATPase and DNA-cleavage activities of several mutants which are catalytically deficient. These mutants include K76R, which is a mutation in the packaging ATPase site, T194M, which packages at a 10-fold slower rate in vivo, and G212S, which stalls during packaging. We characterize protomer self-assembly into the tetrameric motor and we compare the catalytic activities of the mutants relative to wild-type. The mechanistic implications of this work are discussed.

Kinetic Analysis of the Genome Packaging Reaction in Bacteriophage λ

Bacteriophage lambda is a double-stranded DNA virus that infects the Escherichia coli bacterium. lambda genomic DNA is replicated via rolling circle replication, resulting in multiple genomes linked head to tail at the cos site. To insert a single lambda genome into the viral capsid, the lambda terminase enzyme introduces symmetric nicks, 12 bp apart, at the cos site, and then promotes a strand separation reaction, releasing the tail end of the previous genome and leaving a binary complex consisting of lambda terminase bound to the head end of the adjacent genome. Next, the genome is translocated into the interior of the capsid particle, in a process that requires ATP hydrolysis by lambda terminase. Even though DNA packaging has been studied extensively, currently no bulk assays are available that have been optimized to report directly on DNA translocation. Rather, these assays are sensitive to assembly steps reflecting formation of the active, DNA packaging machine. In this work, we have modified the DNase protection assay commonly used to study DNA packaging in several bacteriophage systems, such that it reports directly on the kinetics of the DNA packaging reaction. We have analyzed our DNA packaging data according to an N-step sequential minimal kinetic model and have estimated an overall packaging rate of 119 +/- 8 bp/s, at 4 degrees C and 1 mM ATP. Furthermore, we have measured an apparent step size for the this reaction (m(obs)) of 410 +/- 150 bp. The magnitude of this value indicates that our assay is most likely sensitive to both mechanical steps associated with DNA insertion as well as occasional slow steps that are repeated every >410 bp. These slow steps may be reflective of the pausing events observed in recent single-molecule studies of DNA packaging in bacteriophage lambda [Fuller, D. N., et al. (2007) J. Mol. Biol. 373, 1113-1122]. Finally, we show that either ATP or ADP is required for terminase cutting at cos, to generate the active, DNA packaging complex.

Single phage T4 DNA packaging motors exhibit large force generation, high velocity, and dynamic variability

Terminase enzyme complexes, which facilitate ATP-driven DNA packaging in phages and in many eukaryotic viruses, constitute a wide and potentially diverse family of molecular motors about which little dynamic or mechanistic information is available. Here we report optical tweezers measurements of single DNA molecule packaging dynamics in phage T4, a large, tailed Escherichia coli virus that is an important model system in molecular biology. We show that a complex is formed between the empty prohead and the large terminase protein (gp17) that can capture and begin packaging a target DNA molecule within a few seconds, thus demonstrating a distinct viral assembly pathway. The motor generates forces >60 pN, similar to those measured with phage phi29, suggesting that high force generation is a common property of viral DNA packaging motors. However, the DNA translocation rate for T4 was strikingly higher than that for phi29, averaging approximately 700 bp/s and ranging up to approximately 2,000 bp/s, consistent with packaging by phage T4 of an enormous, 171-kb genome in <10 min during viral infection and implying high ATP turnover rates of >300 s(-1). The motor velocity decreased with applied load but averaged 320 bp/s at 45 pN, indicating very high power generation. Interestingly, the motor also exhibited large dynamic changes in velocity, suggesting that it can assume multiple active conformational states gearing different translocation rates. This capability, in addition to the reversible pausing and slipping capabilities that were observed, may allow phage T4 to coordinate DNA packaging with other ongoing processes, including viral DNA transcription, recombination, and repair.

Quantification of the DNA cleavage and packaging proteins U(L)15 and U(L)28 in A and B capsids of herpes simplex virus type 1.

The proteins produced by the herpes simplex virus type 1 (HSV-1) genes U(L)15 and U(L)28 are believed to form part of the terminase enzyme, a protein complex essential for the cleavage of newly synthesized, concatameric herpesvirus DNA and the packaging of the resultant genome lengths into preformed capsids. This work describes the purification of recombinant forms of pU(L)15 and pU(L)28, which allowed the calculation of the average number of copies of each protein in A and B capsids and in capsids lacking the putative portal encoded by U(L)6. On average, 1.0 (+/-0.29 [standard deviation]) copies of pU(L)15 and 2.4 (+/-0.97) copies of pU(L)28 were present in B capsids, 1.2 (+/-0.72) copies of pU(L)15 and 1.5 (+/-0.86) copies of pU(L)28 were found in mutant capsids lacking the putative portal protein pU(L)6, and approximately 12.0 (+/-5.63) copies of pU(L)15 and 0.6 (+/-0.32) copies of pU(L)28 were present in each A capsid. These results suggest that the packaging machine is partly comprised of approximately 12 copies of pU(L)15, as found in A capsids, with wild-type B and mutant U(L)6(-) capsids containing an incomplete complement of cleavage and packaging proteins. These results are consistent with observations that B capsids form by default in the absence of packaging machinery in vitro and in vivo. In contrast, A capsids may be the result of initiated but aborted attempts at DNA packaging, resulting in the retention of at least part of the DNA packaging machinery.

Functional Analysis of the Terminase Large Subunit, G2P, of Bacillus subtilis Bacteriophage SPP1

The terminase of bacteriophage SPP1, constituted by a large (G2P) and a small (G1P) subunit, is essential for the initiation of DNA packaging. A hexa-histidine G2P (H6-G2P), which is functional in vivo, possesses endonuclease, ATPase, and double-stranded DNA binding activities. H6-G2P introduces a cut with preference at the 5'-RCGG downward arrowCW-3' sequence. Distamycin A, which is a minor groove binder that mimics the architectural structure generated by G1P at pac, enhances the specific cut at both bona fide 5'-CTATTGCGG downward arrowC-3' sequences within pacC of SPP1 and SF6 phages. H6-G2P hydrolyzes rATP or dATP to the corresponding rADP or dADP and P(i). H6-G2P interacts with two discrete G1P domains (I and II). Full-length G1P and G1PDeltaN62 (lacking domain I) stimulate 3.5- and 1.9-fold, respectively, the ATPase activity of H6-G2P. The results presented suggest that a DNA structure, artificially promoted by distamycin A or facilitated by the assembly of G1P at pacL and/or pacR, stimulates H6-G2P cleavage at both target sites within pacC. In the presence of two G1P decamers per H6-G2P monomer, the H6-G2P endonuclease is repressed, and the ATPase activity stimulated. Based on these results, we propose a model that can account for the role of terminase in headful packaging.

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.

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 (gpA 1·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 (gpNu1 SRK) (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 (gpNu1 GDN). The circular dichroism (CD) spectra of both protein preparations suggest that they are predominantly α-helical when purified and stored in Tris buffers. Moreover, thermal denaturation of the proteins thus purified yielded similar thermodynamic parameters for unfolding ( T m, Δ H m and Δ S m of unfolding of ≈306 K, ≈22 kcal/mol and ≈70 cal/mol·K, respectively). Interestingly, however, when the proteins were purified and stored in imidazole buffers, the gpNu1 SRK preparation lost a significant amount of secondary structure and was more stable to both thermally-induced and guanidinium HCl-induced denaturation than was gpNu1 GDN. 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 gpNu1 SRK and gpNu1 GDN 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.

Characterization of the Small Subunit of the Terminase Enzyme of theBacillus subtilisBacteriophage SPP1

The small subunit of bacteriophages SPP1 and SF6 terminase, G1P, share 71% identity clustered in three conserved segments (I, II, and III). Within segment I the helix–turn–helix DNA-binding domain was mapped, whereas segment III was found to be nonessential. For terminase activity, chimeric G1Ps, obtained by domain swapping between gene1of SPP1 and the SF6 origin (Chi1 to Chi4), were purified. The chimeric proteins behave in all respects similarly to the G1P of SPP1 or SF6. The major determinant for G1P:G1P interactions was found to lie within segment II. We showed that a G1P derivative (G1P*) lacking the 62 N-terminal residues (segment I), and Chi1 lacking the 45 C-terminal residues (segment III) interact with G1P. The N-terminal domain of G1P is necessary for terminase subunit assembly, because the large subunit of the terminase (G2P) interacts only with G1P and Chi1, but fails to do so with G1P*. These results suggest that segment III and the extended C-terminal part of SPP1 G1P do not play a major role in DNA recognition and that G1P recognizes an extended nucleotide sequence and DNA structure.

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.