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Originally published In Press as doi:10.1074/jbc.M602093200 on June 28, 2006

J. Biol. Chem., Vol. 281, Issue 35, 25635-25643, September 1, 2006
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Mechanistic Coupling of Bacteriophage T4 DNA Packaging to Components of the Replication-dependent Late Transcription Machinery*

Lindsay W. Black1 and Guihong Peng

From the Department of Biochemistry and Molecular Biology, University of Maryland Medical School, Baltimore, Maryland 21201-1503

Received for publication, March 6, 2006 , and in revised form, June 5, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Regulation of the terminal stage of viral DNA development, DNA packaging, is poorly understood. A new phage T4 in vitro DNA packaging assay employed purified proheads, terminase (gp17 + gp16), and ATP to encapsidate DNA resistant to nuclease. Mature phage T4 DNA and linearized plasmid DNAs containing or lacking a cloned T4 gene were packaged with high (~10%) efficiency. Supercoiled, relaxed covalently closed, and nicked circular plasmid DNAs were packaged inefficiently, if at all, by these components. However, efficient packaging is achieved for nicked circular plasmid DNA, but not covalently closed plasmid DNA, upon addition to packaging mixtures of the purified T4 late transcription-replication machinery proteins: gp45 (sliding clamp), gp44/gp62 (clamp loader complex), gp55 (late {sigma}-factor), and gp33 (transcriptional co-activator). The small terminase subunit (gp16) is inhibitory for packaging linear DNAs, but enhances the transcription-replication protein packaging of nicked plasmid DNA. Taken together with genetic and biochemical evidence of a requirement for gp55 for concatemer packaging to assemble active wild-type phage particles (1), the plasmid packaging results show that initiation of phage T4 packaging on "endless" concatemeric DNA in vivo by terminase depends upon interaction with the DNA loaded gp45 coupled late transcription-replication machinery. The results suggest a close mechanistic connection in vivo between DNA packaging and developmentally concurrent replication-dependent late transcription.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Phage T4 replicates its DNA autonomously with a full set of phage-encoded replication proteins (2). Replication culminates in many fold amplification of injected linear ~170-kb mature phage DNA to form concatemeric DNA. This "endless" DNA is the product of multiple replication and recombination pathways (3). Concatemeric DNA is the in vivo substrate for DNA packaging into proheads and for the generation of mature phage DNA by terminase (4).

Phage T4 employs Escherichia coli RNA polymerase to carry out a complex program of early, middle and late transcription by synthesizing numerous proteins that modify RNA polymerase (RNAP)2 promoter recognition. In the late stage of its development, concurrent T4 phage DNA replication and late transcription are intimately connected (5). A unique mechanism accounts for the following interesting features of late phage T4 development: phage T4 late transcription depends upon: (i) concurrent DNA replication; (ii) activation of RNAP by late transcription proteins (gp55, late {sigma}-factor; gp33, transcription co-activator); and (iii) direct action of the DNA polymerase processivity factor gp45. The mechanism of late transcription at short (-10 TATAAATA, no -35) late T4 promoters has been shown to require tracking along the DNA of the gp45 sliding clamp to which gp55 and gp33 attach and through which contact is made with RNAP (6). In fact, these two transcription factors and DNA polymerase (gp43) share similar C-terminal peptide sequences that account for gp45 binding (7). In addition, late transcription in vitro is shown to be activated by components of the replication fork machinery (6, 8). The sliding clamp is loaded onto the DNA at a nick or single-stranded region of replicating DNA by the clamp loader complex (gp44/gp62) (9). Thus the sliding clamp has been demonstrated to serve a dual replication-transcription function (for an overview of replication and late transcription, see "Discussion").

Packaging of concatemeric phage T4 DNA proceeds concurrently with replication and late transcription and follows a mechanism generally conserved among many bacteriophages (10). Mature T4 DNA can be packaged in vitro into proheads by terminase in a simple system that bypasses DNA end formation (11). However, although initiation of phage T4 DNA packaging on concatemers in vivo remains to be understood, it can be efficient in vitro (1, 12). There is evidence that a gene 16 pac site participates in this process and that gp16 binds preferentially to this site when a gp16 ring complex is renatured with DNA (13, 14). The phage T4 DNA packaging enzyme terminase is a multimeric enzyme composed of multiple copies of large (gp17, 70 kDa) and small subunits (gp16, 17.5 kDa) (15, 13). The large subunit carries out the nuclease or "terminase" processing of the DNA concatemer, although this activity must be tightly controlled since in vitro nuclease activity of gp17 has not been reported (16, 17, 19). The large terminase subunit also contains a high turnover ATPase activity that likely translocates DNA into the prohead (12, 17, 18). The small subunit stimulates ~100-fold the high activity gp17 ATPase (12, 19). Relatively weak gp16 binding to gp17 multimerizes gp17 and following multimerization, the high turnover ATPase does not appear to require continued gp16 interaction (19). The terminase large subunit has been known to interact with a number of phage T4 proteins that include the portal protein of the prohead (gp20), gp32 (single strand DNA-binding protein), the small terminase subunit (gp16) as well as itself (gp17) (summarized in Ref. 1). Phage display identified these previously identified terminase interacting proteins, and moreover, addition of gp32 and of gp20 to gp17 led to an enhancement of its ATPase activity, in support of the biological significance of these protein interactions (1, 19).

Bipartite dispensable capsid protein HOC (highly antigenic outer capsid protein) and SOC (small outer capsid protein) phage T4 display of randomized peptides led also to the unexpected observation that the large terminase subunit (gp17) interacted with the phage late {sigma}-factor (gp55) (1). A number of lines of evidence supported the gp17-gp55 interaction that was inferred from displayed amino acid sequence matches to a single peptide region of gp55: (i) gp17-gp55 co-immunoprecipitation; (ii) gp17-gp55 co-elution by affinity column chromatography; and (iii) gp17 binding to a column-coupled peptide corresponding to the display identified residues of gp55. A packaging role of this interaction was suggested by inhibition by the identified gp55 peptide of in vitro DNA packaging to form viable phage. Moreover, concatemeric DNA made by a T4 mutant with inactivated genes 55 and 33 was inert for packaging as compared with DNA concatemers produced by T4 prohead mutants (e.g. gene 23) that synthesized gp55 and gp33. These observations suggested a possible direct role of gp55 and other components of the late transcription apparatus in initiating packaging on concatemers. However, the 55--33- packaging deficiency could also be an indirect consequence of changes in concatemer structure; e.g. a more branched concatemer is known to accumulate in the absence of endonuclease VII synthesis in a 55--33--deficient infection (1). These complexities suggested the need for a more direct test of a gp55 role in packaging with simpler and better defined DNA substrates and packaging components, as well as an assay that does not require infective phage assembly.

In this work a new defined in vitro DNA packaging system is described that employs only purified terminase and T4 proheads to package phage and plasmid DNAs. Packaging is assayed by a nuclease DNA protection assay rather than by active virus formation (11), and it is shown that packaging displays comparable requirements in both assays. Whereas packaging of linear DNAs can be carried out efficiently by terminase alone in this assay as previously reported for packaging of mature phage T4 and other phage and plasmid DNAs with the phage assembly assay (15, 12, 20, 21), packaging of circular plasmid DNAs is highly inefficient. Addition of several T4 proteins of the replication-late transcription apparatus together with the small terminase subunit is shown to stimulate packaging of such DNAs to levels approaching linear DNAs. It is probable that these requirements mimic those for the packaging of other DNA molecules without ends, including T4 concatemers, the in vivo substrate for DNA packaging.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
DNAs—Phage T4 DNA (~170 kb) was purified from CsCl-purified phage particles as previously described (22). Plasmid DNAs pET12 (4674 bp) and pL16 (pET12 containing a 690-bp T4 DNA fragment that included gene 16) (4845 bp) were previously described in T4 transduction experiments that showed enhanced packaging in vivo of the apparent gene 16 pac site containing plasmid compared with the parent plasmid (13, 14). Plasmid DNAs were purified by a standard alkaline lysis procedure followed by silica gel-based purification (Denville Scientific or Qiagen) (23). Relaxed circular plasmid DNAs were prepared by topoisomerase 1 incubation (Fermentas). Nicked plasmid DNAs were prepared by incubation of pET12 and pL16 with the nicking enzyme Nb.Mva1269I (Fermentas) that is limited to one nicking site (GAATGC) in these plasmid DNAs and that produced no linearized plasmid DNA. Alternatively, as noted in some experiments, the nicking enzyme Nb.Bpu10I (Fermentas) generated nicked circular DNA at a single nicking site (GCTNAGC) in these plasmids together with ~3-5% linearized DNA. Fully linearized plasmid DNAs were produced by PstI restriction endonuclease digestion. Total packaging DNA substrate concentrations were determined by A260 as confirmed by SYBR Gold staining agarose gel electrophoresis followed by quantitation of agarose gel DNA bands. DNA processing by commercial enzymes was according to the manufacturers' instructions.

Packaging Proteins—Unmodified and untagged terminase proteins gp17 and gp16 were purified from expression vectors as previously described (19, 13). Terminase was constituted by mixing gp16 (30 mg/ml) to 17.2 µM together with gp17 (0.73 mg/ml) to 10.4 µM immediately before use. Proteins gp45 and His-tagged gp55 were purified as described from expression vectors provided by Geiduschek and co-workers (24) and His-tagged gp33 and gp44/gp62 were generous gifts of Geiduschek and co-workers and prepared as previously described. All protein concentrations were determined by Bradford protein assay and the purity of proteins was estimated by SDS-PAGE followed by Coomassie Blue and silver staining as described under "Results."

Proheads—Proheads were purified by differential centrifugation of extracts from E. coli P301 infected for 2 h at 20°C with 16 amN66-17 amA465-13 amE111-rIIA({Delta}H88) as described (25). The mutant infection accumulates proheads because of terminase deficiency (16 am-17 am), lacks the prohead neck (13 am) to prevent premature prohead-tail joining, and contains the rIIA ({Delta}H88) mutation to allow assay of phage formation by T4 wild-type DNA addition to the packaging mixture (11). The concentrated and partially purified proheads were loaded on a 15-45% glycerol gradient prepared in buffer A (50 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 0.5 mM EDTA). Following centrifugation for 2 h at 35,000 rpm in a Beckman SW 50.1 rotor at 4 °C, a relatively sharp prohead band that was visualized ~three-fourths of the distance to the bottom of the tube was removed by side puncture. The glycerol gradient-purified proheads were then chromatographed by a linear gradient of NaCl to 0.5 M in buffer A on an FPLC DEAE column run at 1 ml/min (100 psi), which resolved three discrete peaks: empty large proheads (elps) (~0.1 M), empty small proheads (esps) (~0.2 M), and nucleic acid (~0. 3 M NaCl). The proheads were concentrated by high speed centrifugation (18,000 rpm in a Sorvall SS34 rotor) and resuspended in buffer A. Prohead concentration was determined by comparison of the quantity of the major capsid protein to that of a known quantity (by titering and A260) of CsCl-purified phage particles judged by SDS-PAGE and Coomassie Blue staining.


Figure 1
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  		FIGURE 1.
Packaging of linear plasmid and mature phage T4 DNAs requires the T4 terminase large subunit gp17, proheads, and ATP by a nuclease protection assay. Lane 1, kb agarose gel DNA markers. Lanes 2-8, PstI-linearized pL16 plasmid DNA packaged with: lane 2, all components; lane 3, omitting elp; lane 4, omitting terminase large subunit gp17; lane 5, with non-hydrolyzable AMP-PCP replacing ATP; lane 6, with terminase (gp17 plus gp16) replacing the large terminase subunit (gp17); lane 7, gp17 rather than terminase; lane 8, with esp replacing elp. Lanes 9-12, mature phage T4 DNA packaged with: lane 9, all components; lane 10, without elp proheads; lane 11, without terminase large subunit gp17; lane 12, with esps rather than elps.

 
In Vitro Packaging (IVP) Assay—Packaging was carried out by mixing 2x RB, water, DNA, T4 proteins mix (for 10 min), proheads, and terminase, added in this order, to a final volume of 16 µl and a final concentration of 1x RB at room temperature, generally for 1 h. Terminase was gp17 and gp16 mixed immediately before the assay. 2x RB contains 100 mM Tris-HCl, pH 8.0, 12 mM MgCl2, 1 mM dithiothreitol, 4 mM spermidine, 10 mM ATP, 200 mM NaCl, 4 mM putrescine, and 10% polyethylene glycol (15-20 kDa, Sigma). Final concentrations of reactants were as follows: 1) DNAs: 0.1-0.3 µg of plasmid DNA (~2-5 nM)or0.3 µg of mature T4 DNA (~0.17 nM) were added to the reaction mixture; 2) proteins: when added to the packaging mixture in 1x RB, the following final protein monomer concentrations were employed: gp17 (1.3 µM), gp16 (2.1 µM), gp45 (0.19 µM), gp55 (0.53 µM), gp33 (0.21 µM), and gp44/gp62 (0.17 µM of the 4:1 complex) (in addition to the clamp loader, several of these proteins are known to multimerize: gp16 ~8-mer and two times ~8-mer, mainly the ~8-mer, gp45, trimer, and gp17, high activity ATPase multimer (~6-mer formed by gp16 interaction); 3) Proheads: either empty small proheads (esps) or empty large proheads (elps) were added to 1.6 x 1010 (1.7 nM). Following the completion of packaging, 1 µl of protease-free pancreatic DNase (Sigma D-4527) (20 mg/ml) was mixed, centrifuged 1 min at 2,000 rpm in a microcentrifuge, and incubated for 30 min at 37 °C. Benzonase (Novagen) was also employed as the nuclease in some trial experiments that yielded comparable results. Following nuclease digestion, 3 µl of a mixture of 1 part proteinase K (Roche Applied Sciences) (20 mg/ml), 1 part EDTA (0.5 M, pH 8.0), and 1 part SDS (10%) was mixed, centrifuged 1 min at 16,000 rpm, and incubated at 65 °C for 30 min. The solution was mixed with 5 µl of 5x glycerol dye-containing running buffer, and electrophoresis was carried out on a 0.8% agarose gel run in Tris-acetate-EDTA. The gel was stained for 30 min with SYBR Gold (Molecular Probes, Inc) and visualized on a UVP fluorescence light box. Packaged DNAs were quantitated using UVP software to integrate DNA band density in comparison to known quantities of a standard 1-kb DNA ladder (Fermentas). AMP-PCP (Sigma M-7510) was employed as non-hydrolyzable ATP inhibitor that replaced ATP in 2x RB.

gp17, gp55, and gp45 Binding Experiments—Purified proteins (Fig. 3) were used in: 1) co-immunoprecipitation experiments carried out essentially as described (19) except that washings of the protein A-Sepharose (Amersham Biosciences) gp17 antiserum precipitate resuspended and incubated with purified protein mixes were carried out in SM buffer, and in 2) affinity column binding experiments employing a gp17-intein-chitin binding domain self-cleaving fusion protein retained on a chitin affinity column as described (1).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Validity of a New in Vitro Phage T4 DNA Packaging Assay for Simple DNA Substrates—A new nuclease protection assay of phage T4 packaging was developed that employs model DNA substrates and purified proteins to assess directly the role of transcription and replication proteins in packaging. Packaging of linear plasmid DNA shows a complete dependence upon phage T4-expanded proheads (elp) and terminase large subunit gp17 (Fig. 1, lanes 2-4). Packaging of mature phage T4 DNA displays the same requirements as packaging of linear plasmid DNAs (lanes 9-11). The small molecule requirements for packaging were found to be comparable to those of the phage assembly assay (1, 11, 15, 22); notably, there was a comparable complete dependence upon ATP hydrolysis as in the phage assembly assay (11), as expected from the putative terminase DNA translocating ATPase, because non-hydrolyzable AMP-PCP blocked packaging (lane 5). Packaging of linearized plasmid DNA was more efficient without the small terminase subunit gp16 (lanes 6 and 7). Formation of protected plasmid DNA increases from none at zero minutes until a plateau is reached beginning at about 25 min at room temperature (Fig. 2). These kinetics appear similar although somewhat more rapid than in the phage assembly assay, which also established that the best packaging was at ~30 °C as employed here (11). In the nuclease protection assay, the amount of protected DNA increased linearly with increasing DNA and increasing prohead concentrations until saturation with respect to these variables was observed (data not shown). Comparable numbers of proheads and plasmid molecules were employed in the assay (see "Experimental Procedures") although multiple plasmid molecules are observed to be packaged per prohead in vitro (21). Overall, these observations, and the fact that the packaging requirements in the nuclease protection assay are comparable to those of the previously established viable phage formation assay, support the new assay as a reliable measure of DNA encapsidation.

Mature, Expanded Proheads (elp) Are More Active than Immature, Unexpanded Proheads (esp) in the Plasmid Packaging Assay—Proheads used in the packaging assay were highly purified from contaminating proteins and nucleic acids by glycerol gradient centrifugation followed by DEAE-FPLC chromatography. The proheads are separated by chromatography into two discrete peaks, esps (empty small proheads) and expanded mature elps (empty large proheads). Although these two separable classes of proheads have distinct sizes and capsid lattice structures, and only elps can bind the dispensable HOC and SOC proteins, they have the same basic protein composition (25-27). It is seen that the highly purified proheads contain only the head subset of the proteins of CsCl-purified whole phage particles (Fig. 3, lane 11). As previously shown, the mature capsid protein gp23 (dot, Fig. 3, lane 11) in elps, as in phage, is unable to enter an SDS-PAGE gel unless heated (lanes 9 and 10), whereas the gp23 in esps enters the gel without heating (Fig. 3, lanes 7 and 8) (25-27). Elps were active in packaging, whereas in several independent preparations the esps were found to be scarcely active in packaging of either plasmid or phage T4 DNAs (e.g. Fig. 1, lane 7 versus 8 and 9 versus 12). Thus the observed lower packaging activity of esp proheads is consistent with, but appears even more pronounced than in the viable phage assay (25). This is of interest because packaging of single ~5-kb plasmid DNA molecules, unlike T4 DNA, is not expected to expand the esp to the elp prohead structure, and thus should not pose an energetic barrier to packaging. Overall, therefore, although it has been concluded from a number of lines of evidence that DNA packaging is generally the triggering event that leads to the esp to elp transformation in vivo (4), evidently in vitro packaging requirements display differences from those in vivo, where it has been concluded that elp proheads are inert for packaging (28).


Figure 2
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  		FIGURE 2.
Packaging of linear plasmid DNA increases with time and plateaus by about 25 min at room temperature. The amount of packaged nuclease-protected pET12 DNA was quantitated by Sybr Gold staining and UVP software analysis of integrated DNA band density; the curve is fitted to the averages of three experiments.

 
Packaging of Linear but Not Circular DNA—Packaging of linearized plasmid DNA showed no significant dependence on the T4 DNA content of the plasmid when tested with the pac containing small terminase gene 16 sequence plasmid (Fig. 4, lanes 1 and 2). Packaging of both linearized plasmid DNAs (pL16 with the 16 gene and the parent plasmid pET12) was measured to be 14% of the input DNA and was more efficient without gp16 (Fig. 4, lanes 3 versus 4, Fig. 1, lanes 6 versus 7, and Fig. 7, lanes 1 versus 2). However, when the pL16 circular plasmid DNAs containing the T4 gene 16 pac sequence were tested, there was little or no packaging of the supercoiled, relaxed, or nicked-relaxed plasmid DNAs by terminase (Fig. 4, lanes 5-8). Similar results were obtained with pET12 DNA and with gp17 (data not shown).

Purification and Binding Properties of T4 Late Transcription and Replication Proteins (LT/RPs)—All of the proteins employed in the nuclease protection and protein interaction assays are displayed in Fig. 3a and they are seen to be ~70-90% pure by SDS-PAGE and Coomassie Blue staining. A number of biochemical tests that are summarized in the introduction including co-immunoprecipitation of gp17 with gp55 antiserum had demonstrated the affinity of gp17 for gp55. Phage display had also suggested the possibility of terminase-gp45 interaction, although the peptide matches were significantly weaker statistically and dispersed throughout gp45 as compared with the single peptide clustered matches to gp55 (1). Therefore new co-immunoprecipitation experiments employing an antiserum against gp17 were carried out to investigate the possibility of gp17 interaction with gp45. However these experiments show that precipitation of gp45 depends upon the presence of gp55 (Fig. 3b, lane 3 versus 4). In agreement with this conclusion, when a mixture of purified gp45+gp55 proteins was passed over a chitin column to which an extract containing gp17-intein-chitin binding domain fusion protein had been adsorbed, binding of gp45+gp55 proteins was evident by co-elution with intein-cleaved gp17 from the column. However, in the absence of gp55, gp45 did not display binding to the gp17 containing column (data not shown). Because it is known that gp55 and gp33 bind to gp45 and exert their transcription effects by means of this binding (7), and our experiments show that binding of gp17 to gp45 appears to require the presence of gp55, it is likely that most or all of the gp17 late transcription/replication protein binding effects observed are caused by the high affinity gp17 binding to gp55 that was demonstrated previously (1).


Figure 3
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  		FIGURE 3.
Characterization by SDS-PAGE of the purity of proteins and proheads employed in the packaging assays (a) and their binding properties (b). a, 1.5-µg aliquots of each purified protein were loaded onto a 14.5% SDS-PAGE gel, which was stained with Coomassie Blue. Lanes 1-11, gp17, gp16, gp45, gp55, gp33, gp62/gp44, esp proheads (unboiled), esp proheads (boiled), elp proheads (unboiled), elp proheads (boiled), boiled purified T4 phage, the dot shows the major capsid protein gp23. Lane 12, a mixture of high and low molecular mass rainbow marker proteins (220, 97, 66, 45, 30K, 20, 14.3 kDa). b, terminase gp17 subunit binding properties of gp45 and gp55 determined by co-immunoprecipitation. Lane 1, gp45 + gp55+ gp17 - gp17 IgG protein A-Sepharose; lane 2, gp55 + gp17 + gp17 IgG protein A-Sepharose; lane 3, gp45 + gp17 + gp17 IgG protein A-Sepharose; lane 4, gp55 + gp45 + gp17 + gp17 IgG protein A-Sepharose. The indicated protein positions on a 14.5% SDS-PAGE gel-stained with Coomassie Blue are displayed following co-immunoprecipitation of ~1 µg of the addition proteins with gp17 IgG protein A-Sepharose as described under "Experimental Procedures."

 


Figure 4
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  		FIGURE 4.
Unsupplemented packaging of linearized plasmid DNAs with or without T4 genes occurs with high efficiency, whereas circular supercoiled, relaxed, or nicked-relaxed DNAs are inefficiently packaged. Lanes 1 and 2, packaging of PstI-linearized pL16 and pET12 DNAs (0.2 µg); lanes 3 and 4, packaging of PstI-linearized pL16 by gp17 and by terminase (0.3 µg of DNAs), and lanes 5-8, packaging of supercoiled, linearized, nicked, and relaxed covalently closed pL16 plasmid DNA (0.1 µg), respectively, by terminase. Lane 9, kb agarose gel DNA markers.

 
Stimulation by LT/RPs of in Vitro Packaging of Circular Plasmid DNA—To characterize the effect of T4 late transcription and replication proteins on packaging, we tested the capacity of purified proteins to stimulate packaging of the circular plasmid DNAs which were otherwise nearly inert in the in vitro packaging mixture. When purified proteins are added to the in vitro packaging assay, the late transcription replication proteins mixture (LT/RP) stimulates packaging of nicked L16 plasmid DNA by ~20-fold (Fig. 5, lanes 2 versus 3 and 8 versus 7). The stimulation by the LT/RP protein mix of packaging of the nicked plasmid DNA is markedly better than that of the supercoiled plasmid DNA (lanes 10 and 11). Removing individual proteins from the stimulatory protein mix reduces the stimulation significantly (lanes 3 and 7 versus 4, 5, and 6). The upper (~10 kb) DNA visualized in packaging of the nicked and other circular plasmid DNAs is apparently caused by packaging of plasmid dimer DNA that is present in the substrate plasmid DNA preparations since it is converted to a single PstI band (Fig. 8). When the packaged DNA is quantitated by adding this packaged ~10-kb dimer band to the packaged monomer band, packaging of the input LT/RP-stimulated nicked plasmid DNA is measured to be ~two-thirds that of the input linearized plasmid (lanes 3 and 7 versus 1 and 9) showing high packaging efficiency. Additional experiments showed greater than 20-fold LT/RP-stimulated packaging of nicked but not supercoiled pET12 plasmid DNA. Overall, comparable LT/RP stimulation was observed in more than six independent experiments.

Packaging Proteins Are Free of Nuclease—It might be supposed that the LT/RP stimulation of nicked plasmid DNA packaging is caused by nuclease contaminants in the protein(s) added, so that the nicked plasmid DNAs are first converted to linearized plasmid DNAs which are then efficiently packaged by terminase. However, the LT/RP mix showed no conversion of supercoiled pL16 DNA to linear DNA when assayed under packaging conditions (same protein concentrations and 1x reaction buffer at room temperature for 1 h) (Fig. 6, lane 1 versus 3). Terminase assayed under the same conditions also showed no nuclease activity (Fig. 6, lane 2), in agreement with a previously reported assay on plasmid DNA (19). In addition, the LT/RP mix showed no conversion of nicked pL16 DNA to linear DNA (Fig. 6, lanes 6 and 7 versus 8 and 9) because no increase is observed in the amount of linearized plasmid DNA (dots) produced at low levels during conversion of pL16 supercoiled DNA (lane 5) to nicked DNA by the Nb.Bpu10I nicking enzyme. Neither the terminase nor the LT/RP protein mix showed significant plasmid DNA nicking activity on pL16 plasmid DNA (Fig. 6, lanes 1 and 2). Moreover, in the absence of proheads, neither terminase nor gp17, assayed together with LT/RPs or in their absence, displayed detectable nicking or cutting of pET12 plasmid DNA under packaging assay conditions and protein concentrations (data not shown). Thus the absence of detectable nuclease activity on the circular packaging substrate DNAs supports a direct role of the T4 LT/RPs in stimulating packaging of the nicked plasmid DNA by terminase rather than through its conversion to a packable linearized DNA derivative. Moreover, these experiments also suggest that the nuclease activity displayed in vivo by the large terminase subunit (16) is tightly controlled and apparently inactive until activated by prohead portal interaction and/or by activation that mimics initiation of packaging.


Figure 5
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  		FIGURE 5.
Efficient packaging in vitro of nicked pL16 plasmid DNA following addition of late transcription replication proteins (LT/RPs). Packaging by terminase of equal amounts of l (linear), n (nicked), or sc (supercoiled) plasmid DNAs is compared with (+) or without (-) supplementing the in vitro packaging mixture with the LT/RP proteins (A, gp55, gp45, gp33, gp44/gp62) mixture or omitting individual proteins from the mixture as shown above lanes 1-11. All of the lanes are from a single gel. Lanes 12-14 display dilutions of kb agarose gel DNA markers for quantitation of the packaged DNAs.

 


Figure 6
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  		FIGURE 6.
LT/RPs and T4 terminase display no plasmid cutting or nicking activities under packaging conditions. Lanes 1-3 show pL16 DNA incubated with LT/RP, terminase, and without proteins, respectively; lane 4, kb agarose gel DNA markers plus PstI-linearized pL16 DNA to show resolution of linear (heavy band); lane 5, pL16 plasmid DNA; lanes 6-9, pL16 plasmid DNA nicked with Nb.Bpu10I, which produces a small fraction of linear DNA (dots); lanes 6 and 7, incubated with LT/RP mixture; lanes 8 and 9, incubated without proteins. Lanes 1-4 are from one gel; lanes 5-9 are from a second gel. The positions of the sc (supercoiled), lin (linear), nr (nicked relaxed), and nd (nicked dimer) plasmid DNAs are marked.

 
Packaging of DNA without Ends Is Enhanced by the Small Terminase Protein—Packaging of linear mature phage T4 DNA does not require the small terminase protein, gp16 (Fig. 1), and in fact, gp16 apparently inhibits the packaging of linear plasmid DNA when compared with packaging by gp17 alone (Fig. 7, lanes 1 and 2, see also Figs. 1 and 4). However, when the LT/RP-stimulated packaging of nicked pL16 plasmid DNA by terminase (gp17 plus gp16) is compared with packaging by the large subunit alone (gp17), a marked stimulation by gp16 is observed (Fig. 7, lanes 3-6). The result is of significance because it suggests a novel essential function of the small subunit in packaging nicked circular and presumably concatemeric DNA in vivo (see "Discussion"). This result also argues against nuclease action of one or more of the LT/RPs or their contaminants as an explanation for the LT/RP stimulation of nicked plasmid packaging, since were this stimulation the result of packaging of nuclease generated linearized plasmid DNA, gp16 addition would be expected to be inhibitory rather than enhance packaging, as is observed.

Packaging of Nicked Circular DNA Does Not Produce a Specific End—When the DNAs packaged into proheads in vitro employing linearized and nicked pL16 plasmid DNA are isolated and compared, it appears that the linear DNA is packaged from the PstI-generated end since the packaged DNA is not cleaved by redigestion by PstI restriction endonuclease (Fig. 8A, lanes 1 and 2). In contrast, the packaged nicked plasmid DNA is converted to a smear upon PstI digestion (Fig. 8A, lanes 3 and 4). These PstI-generated smears also arise from packaged nicked dimer (lanes 5 and 6) and show that 16 pac sequences in the plasmid do not lead predominantly to sequence specific packaging cuts. Thus it appears that the DNA ends generated in the LT/RP-stimulated packaging of nicked plasmid DNA are not sequence-specific despite the sequence-specific gp45 loading nick expected at nucleotide 1892, which is located ~2 kb from the PstI site in the pET12 DNA.

Also shown in Fig. 8B is that the LT/RPs have little if any effect on packaging of linearized pL16 DNA (lanes 8 and 9), and display a significant stimulation of packaging of nicked pL16 DNA (lanes 10 and 11), whereas omission of the clamp loader complex (gp44/62) depresses this stimulation (lane 12). Residual stimulation in the absence of the clamp loader complex may not be unexpected because in the presence of PEG addition at 10-5% as in the packaging assay, it is known that gp45 loading onto DNA begins to bypass its clamp loader dependence (29).

The observation that packaging of nicked plasmid DNAs is not associated with introduction of a sequence specific end is compatible with a gp55-gp45 sliding clamp mediated effect of LT/RP proteins on packaging of nicked plasmid DNA, if the mechanism is comparable to their role in late transcription. It is known that once loaded at a nick, the gp55-gp33-clamp can track along the DNA to exert an effect at some distance from the nick at the site of a late T4 promoter (6).


Figure 7
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  		FIGURE 7.
LT/RP stimulation of nicked plasmid DNA packaging enhanced by the small terminase subunit, gp16. Linearized pL16 DNA is more poorly packaged by terminase than by gp17 (lanes 1 and 2) whereas nicked pL16 DNA packaging by terminase is stimulated by LT/RP addition (lanes 3 and 4), whereas packaging by gp17 plus LT/RP addition is weak (lanes 5 and 6). Lane 7, kb agarose gel markers.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The new phage T4 in vitro packaging assay reported here has roughly the same measured efficiency (~10%) of packaging linear plasmid DNA as previous T4 DNA concatemer packaging into viable phages by high levels of terminase-supplemented infected extracts (1). However, the limited number of highly purified proteins and the capacity to employ model substrate DNAs in the assay developed here make it useful for future mechanistic studies of the DNA packaging mechanism. Use of this assay shows a ~20-fold stimulation of in vitro packaging of nicked circular plasmid DNA by purified components of replication coupled late transcription. The biochemical experiments reported here are in good agreement with previous less direct genetic and viable phage packaging work that is summarized in the introduction that suggested a direct gp55 role in concatemer packaging (1). In addition, the biochemical experiments with purified proteins reported here suggest that T4 terminase is loaded onto plasmid DNAs and activated for packaging by interaction with prior loaded gp55·gp45·gp33 ring complexes. The additional involvement of gp45 in the mechanism proposed here is not unexpected in view of the well established late transcription activating properties of gp55 bound to gp45 (6, 30). This suggested mechanism of packaging activation of terminase on phage T4 concatemers by interaction with prior loaded gp55·gp45 ring complexes is also supported by the analysis that suggests highly efficient stimulation of nicked plasmid packaging by the LT/RP proteins is significantly reduced by omission of individual proteins from the LT/RP mixture (Fig. 5). Such a mechanism connects the proteins of three concurrent DNA processes occurring on DNA concatemers late in T4 development (Fig. 9). As summarized in this overview, gp45 is known to be central to both the replication fork and to late replication coupled transcription (2, 6, 30). It appears additionally from our work here that phage T4 diverts the gp55·gp45 complex in part to a DNA packaging role to initiate concatemer packaging.


Figure 8
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  		FIGURE 8.
Packaged DNA isolated from PstI-linearized pL16 DNA has a unique end, whereas LT/RP-stimulated packaged nicked pL16 DNAs does not. A, lanes 1 and 2 show isolated packaged PstI restriction endonuclease linearized pL16 DNA is not detectably cut by PstI redigestion, showing the DNA has a unique PstI end. In lanes 3 and 4 PstI digestion of the isolated LT/RP-stimulated packaged nicked pL16 monomer produces a smear upon PstI digestion; and digestion of the packaged dimer (lanes 5 and 6) also produces a DNA smear in addition to the monomer, indicating packaging of predominantly non-sequence-specific DNA ends. Lane 7,kb DNA agarose gel markers. B, lanes 8 and 9 show that addition of LT/RPs does not significantly affect packaging of linearized pL16 DNA, whereas packaging of nicked pL16 DNA requires the LT/RPs stimulation (lanes 10 versus 11), and is significantly reduced by omitting the clamp loader complex (44/62) (lane 12).

 


Figure 9
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  		FIGURE 9.
Schematic of concurrent late phage T4 packaging (A), transcription (B), and replication (C), emphasizing proteins shared by these DNA transactions and used in these packaging experiments. The phage T4 proteins used are identified below the schematic by gene number and function. The sliding clamp gp45 functions are depicted in the diagram following loading at a DNA nick in three alternative functional modes on branched concatemeric T4 DNA nodes. The double-headed arrow represents concatemer packaging initiated by gp17-gp55 interaction that brings together terminase (gp16+gp17) and the transcription factor loaded sliding clamp (gp45+gp55+gp33). Note that in processive headfilling only the initiating first headful packaging event generates a free dsDNA end in the concatemer that may require repair. The diagram shows protein interactions established by the current as well as previous work (2, 30, 1).

 
The dependence upon gp16 to package circular plasmid DNA but not linear DNA is of significance because previously the only well defined biochemical function of gp16 in packaging was the high level stimulation of gp17 ATPase by gp16 (12, 19). This activation apparently proceeds by means of gp17 multimer formation, likely gp17 ring formation induced by gp17 binding to the gp16 ring complex. Following this interaction, the gp17-activated ATPase does not depend upon continuous gp17 gp16 binding because the highly active ATPase is found to be in high molecular weight gp17 complexes without gp16 (19). From this work as well as previous work with the viable phage assembly assay, it appears that gp17 can bind a DNA end to package. However, as purified from expression vectors, neither gp16 nor gp17 displays appreciable binding to single-stranded or double-stranded DNA, and in fact, passage of gp17 through a column-bound mixture of such DNAs removes nuclease and other contaminants, thus terminase binding to DNA via binding to the DNA-loaded gp45 clamp provides a mechanism to circumvent this deficiency (19).

Despite packaging of linear DNA in the absence of gp16 using the current assay and in the previous reports employing the phage assembly assay (12, 13, 15), gene 16 single or double amber mutant deficiency in vivo leads to nearly complete lack of phage formation, although there is slow and inefficient packaging in vivo that likely utilizes alternate terminase defective modes of DNA end formation in the concatemer (4). These properties of gene 16 mutants fit well with the current plasmid DNA findings in suggesting an important role of gp16 in initiating packaging on DNA concatemers. In fact, such a role of the small subunit appears to be a generally conserved feature among two subunit phage terminases, where binding to a cos or pac site in concatemeric DNA, although often dependent upon additional components, strictly requires the small subunit and initiates processive headful DNA packaging, which is subsequently followed by small subunit independent packaging initiated at a DNA end (31, 32). In fact, there is evidence suggesting that T4 DNA packaging occurs preferentially at a gene 16 situated pac site in vivo, a location in or near the small subunit structural gene that is commonly found among many pac and cos site phages (Refs. 14, 31, 32, reviewed in Ref. 10). Moreover, gp16 apparently binds preferentially to gene 16 sequence pac site DNA when renatured from the ring structure with the DNA (13), and gp16 is implicated genetically in DNA binding to this site by its role in a terminase gene amplification involving this site (33). However, nicked plasmid DNAs containing this sequence (pL16) or lacking it (pET12) are not significantly different with respect to packaging in the present assay, nor does packaging of the 16 pac sequence containing DNA lead to sequence specific cutting of the packaged plasmid (Fig. 8). How can this be reconciled with a gp16 role in nicked plasmid DNA packaging? A previous proposal (4) is that as synthesized the gp16 monomer binds to its structural gene, but as the gp16 ring is formed by continued monomer binding, DNA affinity is lost, and gp16 can slide along the DNA. If added to an in vitro packaging mix as the gp16 ring complex, the DNA binding specificity of gp16 may be largely lost, but the gp16-gp17 interaction may still stimulate the initial cutting and packaging of the concatemer. Interaction of gp17 with the loaded LT/RP proteins, possibly a gp55·gp45 ring complex, appears to be a major requirement for this packaging initiation step, and gp16 may participate or subsequently bind to gp17 to promote cutting and packaging. It should be noted that LT/RP stimulation of packaging is not necessarily effected simply by direct terminase-LT/RP interactions, but could additionally involve LT/RP promoted changes to DNA structure that lead to terminase DNA directed activity. Thus in phage {lambda} changes in cos site DNA structure by {lambda} Nu1 and IHF are thought to promote terminase packaging initiation (32), and in phage T4 transcriptional activity on plasmid DNA leads by an unknown mechanism to DNA cutting by terminase in vivo (34).

In addition to promoting DNA concatemer binding to initiate packaging, the terminase gp55·gp45 DNA ring complex interaction mechanism may provide a number of potential advantages for phage T4 late development and regulation. Because gp55 and gp33 bind to the gp45 sliding clamp by C-terminal peptide sequences shared by gp43 (DNA polymerase) (7, 30), there is apparently a potential competition between late transcription and replication at the level of competition for gp45. Similarly, gp17 binding to gp55 might be expected to divert the gp55 loaded sliding clamp from a role in transcription to one in packaging. However no effect of a gene 17 amber mutant on a number of late transcripts' abundance was detected in vivo (1), although direct in vitro measurement of terminase inhibition of synthesis of specific late transcripts may be necessary to exclude a regulatory role in feedback control of transcription. Alternatively, or in addition, it might be desirable for the gp45 sliding clamp to be situated proximal to the cut-packaging site following terminase initiation of processive headful packaging on the concatemer. This could promote DNA repair synthesis and/or recombination into the concatemer of the free DNA end that is not packaged into the first head (Fig. 9). Such a mechanism coupling gp55·gp45 to DNA packaging repair would be analogous to the role of the T7 RNA polymerase in DNA packaging coupled DNA synthesis (35, 36). Further work is necessary to substantiate such a hypothetical DNA repair mechanism that is compatible with the densely interconnected late transcription, replication, and packaging pathways.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant AI11676 (to L. W. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Maryland Medical School, Rm 408, 108 N. Greene St., Baltimore, MD 21201-1503. Tel.: 410-706-3510; Fax: 410-706-8297; E-mail: lblack{at}umaryland.edu.

2 The abbreviations used are: RNAP, RNA polymerase; LT/RPs, late transcription and replication proteins; am, amber mutant; dsDNA, double-stranded DNA; ssDNA, single-stranded DNA; HOC, highly antigenic outer capsid protein; SOC, small outer capsid protein; esps, empty small proheads; elps, empty large proheads; AMP-PCP, beta,{gamma}-methylene adenosine 5'-triphosphate. Back


   ACKNOWLEDGMENTS
 
We thank Peter Geiduschek, Scot Kolesky, Mohamed Ouhammouch, and Sergei Nechaev for their most generous provision of materials and helpful advice. We thank Peter Geiduschek, Julienne Mullaney, Mark Oram, and Venigalla Rao for helpful comments on the manuscript. We thank Fang Liu for assistance in purifying gp45, gp16, and other proteins. We thank Fermentas Inc for providing the nicking enzyme Nb.Mva1269I before commercialization.



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 ABSTRACT
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 DISCUSSION
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