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• W2074955427 abstract "The terminase holoenzyme of bacteriophage λ is a multifunctional protein composed of two subunits, gpNu1 and gpA. In vitro, under certain conditions, terminase can render DNAs from various sources, of varying lengths and termini, resistant to degradation by high concentrations of DNase I. This reaction is completely dependent on the presence of terminase, proheads, a hydrolyzable triphosphate, and a divalent metal ion, and we propose that it is the result of translocation of DNA into proheads by terminase. This reaction is stoichiometric with respect to terminase, DNA, and proheads and can be supported by all deoxyribo- and ribonucleoside triphosphates, but not by the corresponding diphosphates or nonhydrolyzable ATP analogs. Mg2+ and Ca2+ promote the reaction, but Mn2+ and Zn2+ do not. In the absence of spermidine, translocase activity is low, but addition of the Escherichia coli protein integration host factor (IHF) promotes specific translocation of only those DNA fragments containing the terminase-binding site, cosB. When spermidine is present, nonspecific translocation of DNA from any source is stimulated. Under these conditions IHF no longer promotes specificity, but translocation of only cosB-containing DNA fragments can be restored by addition of small amounts of a dialyzed and RNase-treated E. coli extract, suggesting that additional host factor(s) may be involved in determination of packaging specificity. To a limited extent, gpA alone can promote translocation, but gpNu1, which has no translocase activity on its own, must be added to approach the holoenzyme-like activity levels. Formation of viable phage cannot be accomplished by gpA in the absence of gpNu1. The terminase holoenzyme of bacteriophage λ is a multifunctional protein composed of two subunits, gpNu1 and gpA. In vitro, under certain conditions, terminase can render DNAs from various sources, of varying lengths and termini, resistant to degradation by high concentrations of DNase I. This reaction is completely dependent on the presence of terminase, proheads, a hydrolyzable triphosphate, and a divalent metal ion, and we propose that it is the result of translocation of DNA into proheads by terminase. This reaction is stoichiometric with respect to terminase, DNA, and proheads and can be supported by all deoxyribo- and ribonucleoside triphosphates, but not by the corresponding diphosphates or nonhydrolyzable ATP analogs. Mg2+ and Ca2+ promote the reaction, but Mn2+ and Zn2+ do not. In the absence of spermidine, translocase activity is low, but addition of the Escherichia coli protein integration host factor (IHF) promotes specific translocation of only those DNA fragments containing the terminase-binding site, cosB. When spermidine is present, nonspecific translocation of DNA from any source is stimulated. Under these conditions IHF no longer promotes specificity, but translocation of only cosB-containing DNA fragments can be restored by addition of small amounts of a dialyzed and RNase-treated E. coli extract, suggesting that additional host factor(s) may be involved in determination of packaging specificity. To a limited extent, gpA alone can promote translocation, but gpNu1, which has no translocase activity on its own, must be added to approach the holoenzyme-like activity levels. Formation of viable phage cannot be accomplished by gpA in the absence of gpNu1. During the assembly of phage λ heads in vivo, linear DNA concatamers are matured by the terminase holoenzyme and then specifically translocated into the preformed protein shells, or proheads, starting from the left end of the genome (reviewed in Feiss (1986); Becker and Murialdo(1990); Murialdo(1991)). This translocation reaction is believed to be accomplished by a similar process in all double-stranded DNA bacteriophages (Casjens, 1985; Black, 1988) and is thought to be driven by ATP hydrolysis, since an ATP requirement exists for all in vitro packaging systems (Earnshaw and Casjens, 1980; Black, 1988). Another essential requirement for DNA packaging is the presence of the functional terminase proteins, which are thought to be directly involved in the translocation process (Becker et al., 1977; Guo et al., 1986; Hamada et al., 1986; Rao and Black, 1988). Specificity of λ DNA maturation and translocation in vivo derives from the interactions of the terminase holoenzyme with the DNA elements of the cohesive end junction (cos site), located between −40 and +160 bp on the λ genome map (Miwa and Matsubara, 1982, 1983; Hohn, 1983; Feiss et al., 1983). Cos contains three distinct regions: cosN (−11 to +11 bp), 1The abbreviations used are: bpbase pair(s)gpgene productsssingle-strandedIHFintegration host factorNTPnucleotide triphosphatePFUplaque-forming unitkbkilobase(s)ATPγSadenosine 5′-O-(thiotriphosphate). a sequence with partial 2-fold rotational symmetry where terminase makes the staggered nicks and separates the 12-base overhangs using an ATP hydrolysis-requiring helicase-like activity (Higgins et al., 1988; Rubinchik et al., 1994b); cosB (+12 to +160 bp), which contains three binding sites, R1, R2, and R3, to which both the small subunit of terminase, gpNu1, and the terminase holoenzyme, specifically bind (Shinder and Gold, 1988; Parris et al., 1994), as well as a high affinity binding site for IHF (Kosturko et al., 1989); and cosQ (−12 to −40 bp), a region involved in completion of DNA translocation into the prohead and the cleavage of the 2nd cos site (Cue and Feiss, 1993). base pair(s) gene product single-stranded integration host factor nucleotide triphosphate plaque-forming unit kilobase(s) adenosine 5′-O-(thiotriphosphate). It is clear that the two subunits of λ terminase, gpA (74 kDa) and gpNu1 (21 kDa), must contain domains which define the numerous activities performed by terminase as part of the overall packaging pathway. We have recently established that both the ATPase and the helicase activities of the terminase holoenzyme can be accounted for by its large subunit, gpA (Parris et al., 1994), and have quantitatively analyzed the reaction parameters of these activities (Rubinchik et al., 1994b). We have attempted to investigate the relationship between the two known ATP hydrolysis-requiring activities of terminase, packaging and helicase, and the observed in vitro ATPase. Our interpretation of the results suggested that the in vitro ATPase of gpA and terminase is predominately associated with the helicase activity of these proteins and not necessarily with their translocation activity. However, this interpretation was questionable since the roles and requirements of the various host proteins, as well as of phage morphogenic components required to complete the phage assembly pathway and generate viable PFUs, were not defined in the in vitro packaging assay. DNA packaging assays measure the formation of viable, infectious phage particles, and this formation consists of a series of steps. After the terminase•DNA•prohead complex is formed, DNA is translocated into the capsid. Thereafter, the phage proteins gpD, gpW, and gpFII must be added before tails can be attached (Perucchetti et al., 1988), and there is evidence that host factor(s) may also be involved. In this study, we have used a defined in vitro assay to look specifically at the translocation of DNA into a preformed λ prohead, in the absence of additional host or phage components, and to quantitatively describe the translocase activity promoted by λ terminase and its subunits. The origins and relevant genotypes of bacterial strains used in this work are indicated in Table 1. Bacterial and phage extracts for use in in vitro translocase and packaging assays were prepared as described previously (Parris et al., 1994).Table I Open table in a new tab [14C]DNA labeled with radioactive thymidine from 159T−(λcI857Sam7)) was purified as follows. Cells were grown at 37°C to a concentration of 2 × 108/ml in 500 ml of Frasers medium (a minimal medium with casamino acids and glycerol) supplemented with 0.15 ml of 1 M CaCl2, 25 μl of 20% MgSO4, and 1.25 ml of thymidine (2 mg/ml). Induction was by heating for 15 min at 45°C followed by rapid cooling to 37°C. The culture was grown for 4 h at 37°C in the presence of 250 μCi of methyl-[14C]thymidine to label the induced phage DNA. The specific activity of [14C]λ DNA was 30,000 counts/min/μg. λ phage, labeled and unlabeled mature λ DNA, and M13 ssDNA were purified by standard methods. T7 DNA was the generous gift of Dr. P. Sadowski, and yeast genomic DNA was generously donated by the members of the Dr. B. Andrews Laboratory. Calf thymus DNA (Type II) was from Sigma. The pWP14 (Parris et al., 1988; Rubinchik et al., 1994a) and pTZ19 (Pharmacia LKB) plasmids were purified using Qiagen P500 columns as described by the manufacturers. Plasmid concatamers (>30 kb) were prepared by ligating EcoRI digests of these plasmids at high DNA concentrations. Escherichia coli tRNA was purchased from Boehringer Mannheim, and wheat germ rRNA was generously donated by Dr. B. G. Lane. Strains, vectors, and methods used to purify gpA, gpNu1, terminase, and IHF for use in this study are described in detail by Parris et al.(1994). λ proheads were prepared from thermally induced 594(λAam32Kam24cI857Sam7) cells disrupted by sonication. This was followed by a high speed centrifugation to pellet the proheads. Subsequently a 40-80% ammonium sulfate cut of this fraction was applied to a Sepharose 6B column. The proheads from this column were applied to an anion-exchange TMAE column and were eluted with approximately 0.5 M NaCl. GpA, gpNu1, terminase, IHF, and proheads used in this work were more than 95% pure based on SDS-polyacrylamide gel electrophoresis-Coomassie Brilliant Blue R stain analysis. GpD was partially purified from induced 594 (λWam403Bam10cI857Sam7) with modifications to previously published methods (Perucchetti et al., 1988). Partially purified gpW was the kind gift of Abraham Haddad. Details of its purification will be published elsewhere. For the in vitro assays, all proteins were first dialyzed against buffer containing 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 10% (v/v) glycerol. Protein concentrations were determined after dialysis, using the Bio-Rad protein assay, according to the manufacturer's instructions. Terminase was assumed to have the Mr of 120,000. Deoxyribonuclease I (DNase I, 1861 units/mg dry weight) was obtained from Worthington Biochemical Corporation. Pyruvate kinase (2 mg/ml) was obtained from Boehringer Mannheim. HinDIII, EcoRI, PstI, and ScaI restriction endonucleases and T4 ligase were obtained from New England Biolabs. Acetylated bovine serum albumin was obtained from New England Biolabs as a 10 mg/ml stock solution. 100 mM dNTP solutions, β,γ-methylene-ATP, and ATPγS were purchased from Boehringer Mannheim. 100 mM NTP solutions were purchased from Pharmacia LKB. ADP was purchased from Sigma and contained less than 0.1% of ATP as determined by chromatographic analysis. [2-14C]ATP and [2-14C]dTTP were obtained from DuPont-NEN. Phosphoenol pyruvate was purchased from Sigma. Reactions were typically carried out in volumes of 10-20 μl and contained 0.3-2 μg of DNA, 1-2 μg of purified proheads, 2 mM MgCl2, 1 mM ATP, and 6% glycerol. Reaction mixtures also contained either 20 mM Tris-HCl, pH 8.5, or 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, and 3 mM spermidine. These sets of conditions were respectively analogous to the relaxed stringency (RS) and the high stringency (HS) conditions of the endonuclease activity of λ terminase (Rubinchik et al., 1994a) and will be henceforth referred to as such. Reaction conditions were varied depending on the experiment, as indicated in figure legends. If required, purified IHF was added to 30 nM. For gpNu1:gpA interaction experiments, the two subunits were mixed on ice in the desired molar ratio (typically, 2 or 3 gpNu1 to 1 gpA) and incubated on ice for 5-10 min. All protein dilutions were made in a buffer containing 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 10% (v/v) glycerol, and 1 mg/ml bovine serum albumin. This buffer was used in control reactions which lacked terminase or its subunits. The reactions were initiated by the addition of DNA and were typically incubated at room temperature for 60 min. Unless stated otherwise, 1 μl of a DNase I solution (10 mg/ml) was then added to each reaction, and incubation continued for an additional 10 min. The reactions were stopped by the addition of 3-6 μl of gel loading buffer, which contained 60% glycerol, 150 mM EDTA, and 0.05% bromphenol blue. The reactions were then either electrophoresed directly, or packaged DNA was released from proheads by adding 2-4 μl of 10% SDS to the reactions and incubating them for 5 min at 65°C. Electrophoresis was carried out in 0.7% agarose gels in 0.04 M Tris acetate and 0.002 M EDTA buffer, pH 7.7, at 12 V/cm for 1-2 h. Subsequently, gels were either stained in 0.01% ethidium bromide solution and photographed, or, if labeled DNA was used, dried and exposed to storage phosphor plates overnight, and scanned by the PhosphorImager instrument from Pharmacia LKB. Dried agarose gels could also be stained with Coomassie Brilliant Blue R for protein analysis. Quantitative data analysis was performed by the GraFit data analysis application (version 2.04, Erithacus Software Ltd.), as described previously (Rubinchik et al., 1994a, 1994b). Specifically, all above-background counts remaining in a lane (including the well) after the DNase I treatment were considered to be the result of translocation-dependent protection. ATPase activity was assayed with [2-14C]ATP added to the translocase reactions to a final concentration of 100 μM. Typically, 2-μl aliquots were taken from the translocase assays at various time points and analyzed by thin layer chromatography, as described previously (Rubinchik et al., 1994b). In vitro packaging assays were performed essentially as described (Parris et al., 1994), with the following modifications. Approximately 0.3 μg of mature λcI857Sam7 DNA was added to a reaction containing either 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 3 mM spermidine HCl, 1 mM ATP, 2 mM MgCl2, 15 mM putrescine, and 7 mM 2-mercaptoethanol (HS), or 20 mM Tris-HCl, pH 8.5, 1 mM ATP, 2 mM MgCl2, 30 nM IHF, and 7 mM 2-mercaptoethanol (RS + IHF). To this was added 1.5 μg of purified proheads and purified terminase to a final concentration of 40 nM. The reaction volume at this point was 20 μl. This phase I reaction was allowed to proceed for 20 min at room temperature. Subsequently, a crude extract or combinations of crude extracts and partially purified proteins were added, to provide gpD, gpW, gpFII, and tails, and the phase II reaction was allowed to proceed for an additional 60 min to complete the mature phage particle. Alternatively, all components of the packaging reaction, with the exception of terminase, could be supplied as crude extracts in a single 60-min reaction. Mature phage particles were titered on QD5003, and the results expressed as PFUs/ml reaction. DNase I sensitivity assays were performed with the following modifications to the above system. After the assembly of phase I reactions (with or without gpD and gpW, as desired), they were incubated for 15 min to allow packaging of the DNA to occur. At this point 2 μg of DNase I were added and reactions left to proceed for another 10 min at room temperature. Appropriate phase II components were added and the reaction incubated for another hour at room temperature. Viable phage were titered as described above. DNA packaging assays with other DNA substrates to test for packaging specificity and infectivity of translocated DNA were performed as the 2-phase assays described above, except that λ DNA was replaced by T7 or supercoiled, linear, or concatameric plasmid DNA of the same concentration. T7 DNA packaging was tested by plating phase II products on QD5003 and DH5 and screening for the appearance of characteristic T7 plaques. Plasmid infectivity was monitored by incubation of a plasmid DNA packaging reaction aliquot (10 μl) with 0.1 ml of an exponential culture of QD5003 in 2 ml of culture broth for 1 h at 37°C, followed by plating on solid agar plates containing ampicillin (50 μg/ml), and by observing growth of ampicillin-resistant colonies. 14C-Labeled mature λ DNA was observed to become DNase I resistant when it was used as a substrate in in vitro packaging reactions (Fig. 1, lane 4). This activity required the presence of phage components necessary for correct prohead assembly, such as gpE, gpB, and gpNu3 (Fig. 1, lanes 5, 6, and 8), but not of any other gene products involved in λ phage morphogenesis (Fig. 1, lanes 9-11). Some activity was detected in lysogen extracts which lacked gpC (Fig. 1, lane 7), but these did not generate viable PFUs (Table 2). Purified λ proheads could successfully substitute for prohead-donating lysogen sonicates (Fig. 1, lane 12) in this assay. The amount of DNA observed to be protected from DNase I was 2 orders of magnitude higher than could be accounted for by the number of PFUs generated (Table 2). The assay was equally or more efficient (with respect to the final yield of protected DNA) when purified proheads and terminase were mixed with mature λ DNA in the absence of other phage or host proteins (Table 2). Protected DNA migrated through the gel in a novel pattern, with a large fraction present in a band, the position of which corresponded to the band of purified proheads as assayed by Coomassie Brilliant Blue R staining (data not shown). DNase I resistance was completely dependent on the presence of ′both proheads and terminase (Fig. 2, lanes 7 and 8), as well as MgCl2 and ATP, which leads us to believe that the resistance is the result of terminase-promoted translocation of DNA into preassembled λ proheads.Table II Open table in a new tab Figure 2:Purified proheads and terminase are both necessary and sufficient to promote DNase I resistance. The translocase assays were carried out in 10-μl reaction volumes and under HS conditions, as described under “Experimental Procedures,” except that DNase I was not added to lanes 1, 3, 5, and 7 after a 60-min incubation. All reactions contained 0.8 μg mature λ DNA, of which 0.1 μg was 14C-labeled. Lanes 3, 4, 7, and 8 contained 150 nM terminase; lanes 5-8 contained 9 nM purified proheads.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Heating the DNase I-treated reactions at 65°C in the presence of 1.5% SDS released the DNA from proheads, which then appeared as a distinct band with a mobility similar to that of mature λ DNA. Due to the limited resolution of DNA fragments larger than 20 kb on agarose gels, we estimate the size of DNA species recovered from the proheads to be between 25 and 48.5 kb. The last estimate, which corresponds to the complete length of the λ genome, is unlikely to be accurate, since the products of the translocation assay did not generate viable PFUs after DNase I treatment, unless complemented with partially purified gpD and gpW (Table 2). DNAs of various origins were tested as substrates for the translocase activity of terminase. The results are summarized in Table 3. In the absence of polyamines and NaCl (RS conditions), translocase activity was very low, as assayed by using a HinDIII λ DNA digest as a substrate (Fig. 3, lane 1). When IHF was added to the RS conditions, translocation of the 23-kb DNA fragment, which contains the left end of the λ chromosome and the cosB site, was preferentially enhanced, while that of all the other λ DNA fragments remained low (Fig. 3, lane 2). Titration experiments indicated that maximum IHF effect was reached when this protein was present in 2-3-fold molar excess over the DNA molecules containing the cos site. When a linearized cosmid pWP14 was used as a substrate under RS plus IHF conditions, it was cleaved at the cos site, and the fragment containing the left end-cosB sequences was preferentially translocated.Table III Open table in a new tab Spermidine, or a combination of spermidine and putrescine, significantly enhanced the nonspecific translocase activity, so that under HS conditions all linear DNA molecules of varying lengths and termini, such as all the fragments of a λ HinDIII digest, monomeric T7 genomes, or plasmid DNA, could be translocated into λ proheads, regardless of the presence of the cos site (Fig. 3, lanes 5, 7, and 9; Table 3). Likewise, eukaryotic DNA, from yeast and calf thymus, was translocated under these conditions. However, only those DNA molecules that contained both the left and the right ends of λ chromosome, such as mature λ chromosomes themselves, or concatameric cosmid DNA, 2To generate infectious λ particles, cosmid concatamers were presumably cleaved at two of the multiple cos sites, such that a linear DNA molecule approximately the length of a mature λ chromosome, and containing mature λ ends, was generated. gave rise to infectious particles, as monitored by the appearance of plaques and ampicillin-resistant colonies, respectively (Table 3). Specificity of translocation in the presence of spermidine or both spermidine and putrescine could not be restored by IHF (Fig. 3, lanes 6-8), but was accomplished by addition of small amounts of dialyzed E. coli extract treated with RNase A (Fig. 4, lanes 3 and 5). Purified gpNu1 subunit was not able to promote translocase activity in the absence of gpA. At high protein concentrations and RS conditions (described under “Experimental Procedures”), the large subunit of terminase gpA was capable of protecting a small amount of DNA from DNase I degradation (Fig. 5). It could not, however, promote formation of viable phage. The translocase activity of gpA was not affected by the presence of IHF and did not occur under HS conditions (described under “Experimental Procedures”). It also did not generate viable phage when used as a first stage in a two-stage packaging assay. When the DNA translocated under RS conditions by gpA was extracted from the proheads by heating in the presence of SDS, it appeared as a smear with no distinct bands visible. Translocation of DNA into proheads appeared to be non-catalytic with respect to the amount of terminase, as assayed by quantifying the reaction yields after 60 min at ambient temperature (Fig. 5). The activity was directly proportional to the concentration of terminase below 150 nM but appeared to decrease at higher terminase levels. To examine the activity profile in more detail, we used two levels of terminase from the linear response range to do translocation time courses, as well as to determine ATPase activity under translocation conditions. As can be seen in Fig. 6, the translocation data points were adequately fitted by the first order rate kinetics in both cases. Table 4 summarizes the relevant parameters of this experiment. Calculated reaction yield values confirm that the reactions were essentially completed after a 60-min incubation. The initial reaction rates and the final yields were directly proportional to the terminase concentrations. Similar time course data were obtained when terminase was incubated at room temperature for 1 h prior to the initiation of the reaction, which suggests that time-dependent inactivation of terminase is not the predominant cause of the first order reaction. The translocase activity may be dependent on the number of translocase-competent terminase•DNA•prohead complexes formed. The number of such complexes was estimated for the two terminase concentrations (Table 4), based on the calculated yields and the previously published results which established that up to 82% of λ genome could be packaged into proheads in the absence of gpD (Sternberg and Weisberg, 1977). The average rate of translocation per complex and per terminase protomer was then determined (Table 4).Table IV Open table in a new tab The translocase activity was also found to be stoichiometric with respect to the reaction concentrations of DNA (Fig. 7A) and proheads (Fig. 7B). That is, the yield was directly proportional to the amount of the component tested up to the point where it saturated one of the other reaction components (Fig. 7B). We propose that the number of translocation-competent complexes formed is determined by the reaction concentrations of terminase, DNA, and proheads, as well as relevant physiological conditions, such as spermidine and IHF. The translocase activity of terminase was optimal at MgCl2 concentrations of between 1 and 5 mM. It could also be supported by similar concentrations of CaCl2, but not by MnCl2 or ZnCl2. Translocase was inactive if ATP was absent from the reaction, or if it was replaced by ADP, or by nonhydrolyzable analogs such as ATPγS and β,γ-methylene-ATP. The activity could also be supported, to a varying extent, by all NTPs and dNTPs, with ATP and dATP being the most efficient, followed closely by CTP. We have investigated the effect of varying the ATP concentration on the translocation reaction (Fig. 8). 50% of maximal activity was reached at ATP concentrations of approximately 60 μM, but when concentrations higher than 1 mM were present the activity decreased sharply. This decrease was determined to be the result of Mg2+ depletion by the nucleotide and could be reversed by increasing the concentration of MgCl2. Under RS plus IHF reaction conditions, combining gpA and gpNu1 produced translocase activity comparable to that of the terminase holoenzyme, albeit somewhat less active on a molar basis (Fig. 5). Activity profiles with respect to substrate specificity, as well as various metals, nucleotides, and inhibitors, were also essentially the same. The optimal subunit ratio was between 2 and 4 gpNu1 molecules to 1 gpA molecule (Fig. 9). The subunit mixture was inactive under HS conditions. Bacteriophage λ in vitro packaging assays are very inefficient, with less than 0.05% of the substrate DNA generating PFUs in our experiments (Table 2). By using 14C-labeled λ DNA as a substrate in these assays, we were able to observe some of it becoming resistant to DNase I degradation. This resistance did not require phage proteins responsible for the maturation of λ heads (gpD, gpW, or gpFII), or the presence of phage tails (Fig. 1). DNase I-resistant DNA migrated in a novel pattern on an agarose gel, presumably as the result of its association with phage morphogenic components. Since this resistance required the presence of preassembled λ proheads, active λ terminase, hydrolyzable NTPs, and, in a purified system, could lead to the formation of viable phage after the DNase I treatment, we believe that it arises as the result of translocation of DNA into the λ proheads. The amount of DNA protected was at least 100-fold higher than expected from the number of PFUs observed (Table 2), suggesting that either the viability of the large number of filled proheads was lost at some subsequent morphogenic step (perhaps addition of the tails), or that the majority of DNA translocated into proheads was in some way inviable to begin with (for example, damaged ssDNA overhangs). Translocation activity was not detected with extracts that lacked gpB or gpNu3, which is consistent with previously reported observations that these mutants cannot synthesize λ preconnectors (Georgopoulos et al., 1983). It was also absent in gpE− extracts, which lack the prohead shell, but do make the BC-preconnectors (Georgopoulos et al., 1983). Interestingly, a significant amount of translocation activity was detected with gpC− extracts (Fig. 1, Table 2), which have been reported to make inviable prohead-like structures containing gpE, gpB, and gpB∗, and partially filled with gpNu3 (Georgopoulos et al., 1983). Therefore, the element of the connector composed of gpB (and/or its derivative, gpB∗) appears to be sufficient for interaction with the terminase during DNA translocation. Next, we demonstrated that purified λ proheads could be used successfully in the place of sonicates of induced lysogens. Under HS conditions, terminase and proheads were both necessary and sufficient for the translocase activity to occur (Fig. 2). Translocase was fully active in the presence of RNase, making it unlikely that RNA molecules play an important role in this process in λ, as they do in λ29 (Guo et al., 1987). Translocation also occurred under RS conditions, although much less efficiently. In the presence of IHF, however, the reaction yields under RS conditions equaled or even surpassed those obtained under HS. Both sets of conditions could be used to assemble mature heads in a two-stage in vitro packaging assay (Table 2), indicating that spermidine was not essential to the translocation process itself, but probably acted to facilitate the formation of the DNA•terminase complexes. Presumably, IHF acts in a similar fashion, its binding to the high affinity site in cosB enhancing gpNu1-mediated terminase interactions with that region, as has been demonstrated in vitro for gpNu1 (Shinder and Gold, 1989). The stimulatory effect was not cumulative, i.e. no additional stimulation occurred if IHF was added to the translocase reactions under HS conditions. A similar observation was made on the effects of spermidine and IHF on the in vitro endonuclease activity of terminase (Rubinchik et al., 1994a). When the products of the translocase assay were denatured in the presence of SDS, the released DNA migrated through the 0.7% agarose gel as a single band with mobility similar to that of mature λ. This implies that most of the length of input DNA was packaged into purified proheads. However, when assayed as a substrate for subsequent production of viable phage in a two-stage in vitro packaging assay, the translocase products were found to be sensitive to DNase I treatment (Table 2), suggesting that the entire λ genome could not be packaged into the prohead in the absence of gpD, in agreement with published results obtained in vivo (Sternberg and Weisberg, 1977). Adding gpD and gpW to the translocation assay prior to the addition of DNase I did indeed render the reaction products almost completely insensitive to degradation, as witness the number of PFUs generated (Table 2). These observations are consistent with the previously described roles for gpD and gpW as proteins required to ensure the packaging of the entire λ genome and the stabilization of the mature λ head (Perucchetti et al., 1988). We have investigated the specificity of the in vitro translocase activity of terminase by utilizing a number of nucleic acids as substrates. Neither supercoiled DNA, ssDNA, nor RNA molecules could be translocated under any conditions tested (Table 3). Under RS conditions and in the presence of IHF, the DNA molecules that contained the λ chromosome left end cosB site were specifically and preferentially translocated (Fig. 3, Table 3). Under HS conditions, however, all linear dsDNA molecules could be translocated, irrespective of origin, composition, or the nature of their termini. Such promiscuous translocase activity in vitro has been previously described in a number of other bacteriophages which utilize DNA elements to ensure specificity of packaging in vivo, such as T3 (Hashimoto and Fujisawa, 1988), T7 (Son and Serwer, 1992), and P22 (Behnisch and Schmieger, 1985). In the case of λ translocation in vitro, the failure of non-linear DNA to serve as a substrate implies that the main requirement is a double-stranded terminus, which apparently can have either 5′ or 3′ ssDNA overhangs or be blunt (Table 3). The presence of the proper mature λ termini must therefore become essential at some step following the initiation of DNA translocation. Experiments with T7 and plasmid DNA have demonstrated that unlike T3, λ cannot infect cells successfully in the absence of the cos site, in agreement with the previous reports that specific interactions of λ tails and the right end of the λ chromosome are necessary for both tail attachment and DNA ejection (Xu and Feiss, 1991) and that both mature termini of the λ chromosome are required for its circularization following its injection into a bacterium. For the λ in vitro translocation system, the loss of DNA specificity was correlated with the presence of spermidine in the reactions (Fig. 3). However, packaging of DNA by λ in vivo is very specific, both in the presence and absence of IHF, with no detectable amounts of non-λ DNA found in purified mature phages. Since bacterial cells can contain high concentrations of spermidine and other polyamines, additional factors must play a role in ensuring the specificity of translocation. We have found that the spermidine-promoted loss of specificity could be overcome by addition of small amounts of dialyzed and RNase-treated E. coli C extract, suggesting that other, as yet unidentified, host factor(s) may be involved in the process and that it or they are likely to be protein in nature (Fig. 4). E. coli C was chosen because it was reported to lack cryptic lambdoid prophage sequences (Murialdo, 1988), which would make it unlikely that the effect on specificity of translocation was due to a background expression of some phage protein. The translocase activity also required a hydrolyzable nucleoside triphosphate and a divalent metal ion. All four NTPs and dNTPs could be utilized with comparable efficiency, although ATP and CTP generated higher initial rates of translocation. These results are consistent with the previously characterized NTPase activity of terminase and gpA, but differ from that of in vitro packaging, where CTP was 100-fold less efficient than ATP, and GTP was completely inactive (Rubinchik et al., 1994b). The translocase activity of terminase reached 50% of maximum at approximately 60 μm of ATP (Fig. 8), a concentration comparable with the Km values of the helicase and ATPase activities of gpA and terminase, but significantly different from the ATP requirement for in vitro packaging (Rubinchik et al., 1994b). Both Mg2+ and Ca2+ supported translocation equally well, while Zn2+ and Mn2+ were inactive. In vitro packaging has a similar profile with divalent metals, but there Ca2+ is 100-fold less efficient than Mg2+ (Rubinchik et al., 1994b). Similar results were obtained with studies of potential inhibitors, where sodium vanadate inhibited packaging significantly more than it did translocase at comparable concentrations. The comparison of activity profiles of in vitro packaging and translocase with nucleotides, metals, and inhibitors supports a previously proposed hypothesis that there is some other step(s) in phage morphogenesis that occurs during in vitro packaging that has a more stringent requirement for ATP and Mg2+, and is more sensitive to inhibition, than the translocase activity (Rubinchik et al., 1994b). This step could involve the activities of gpW, gpFII, or tails. Previously, we have reported that purified λ terminase subunits gpNu1 and gpA could be combined to generate in vitro endonuclease, ATPase, and packaging activities resembling those of the terminase holoenzyme (Parris et al., 1994; Rubinchik et al., 1994a, 1994b). A similar result was obtained for the translocase activity. The mixture of the two subunits was less active than the holoenzyme on a molar basis (Fig. 5) but had essentially the same activity parameters with respect to nucleotides, metals, and inhibitors. Unlike terminase, a gpA-gpNu1 mixture was inactive under HS conditions, as was previously reported for its endonuclease activity (Rubinchik et al., 1994a). In both cases, inactivation was correlated primarily with the presence of NaCl in the reaction. The optimal gpNu1 to gpA molar ratio for the translocase activity was between 3 and 4 (Fig. 9), which is higher than the previously reported ratio for the endonuclease activity (between 1 and 2, Rubinchik et al., 1994a). It is possible that translocation of DNA requires a higher degree of occupancy of R sites or another gpNu1 function not related to its DNA binding activity. An intriguing possibility is that the number of gpNu1 molecules associated with each gpA within the terminase protomer actually varies depending on the function being performed, with more gpNu1 being “recruited” as needed. Such an arrangement would be consistent with the non-integer gpNu1/gpA ratios that were previously reported for purified terminase (Gold and Becker, 1983; Tomka and Catalano, 1993a; Parris et al., 1994). Not only the initial reaction rates, but also the final yields of the translocase activity were directly proportional to the reaction concentrations of terminase (Fig. 6, Table 4). The yields were also proportional to the reaction concentrations of DNA (Fig. 7A) and proheads (Fig. 7B). These observations are consistent with the model where ternary complexes are first assembled, the number of such complexes depending on the final concentrations of individual components, as well as the reaction conditions. Titrations of one of these components with the other two being in excess indicate that only a fraction of each of them appears to be capable of forming translocation-competent complexes. In the case of terminase, the amount of inactive protein may be less than it appears, since the number of terminase protomers required per active translocating complex is unknown and could be quite high. It is also possible that components damaged during purification inhibit the reaction by interacting with their active counterparts to form inactive complexes. Translocation of DNA into proheads appears to proceed at a high initial rate, which gradually slows down (Fig. 6). This reaction rate is an average of the complex population and does not tell us what is happening on the level of the individual translocating complex. Since the in vitro translocation reaction does not appear to be processive with respect to terminase (Fig. 5), it is possible that the terminase complex is unable to dissociate from the filled prohead on its own and requires activity of subsequent morphogenic components, such as gpW, gpFII, or λ tails. We are currently investigating these interactions. Terminase has an in vitro NTPase activity which is independent of the translocation reaction (Gold and Becker, 1983; Tomka and Catalano, 1993b; Rubinchik et al., 1994b), while purified λ proheads have no detectable NTPase of their own. When ATPase of the translocation reaction was measured, it was found to be essentially the same as that of terminase and DNA alone (Table 4). The average rate of ATP hydrolysis was sufficient to account for the calculated average rates of translocation, based on the assumption that 2 bp of DNA could be translocated per ATP molecule hydrolyzed (Guo et al., 1987; Morita et al., 1993)." @default.
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• W2074955427 title "The in Vitro Translocase Activity of λ Terminase and Its Subunits" @default.
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