Department of Biology, University of Victoria, PO Box 3020, Victoria, British Columbia V8W 3N5, Canada1
Author for correspondence: David Levin. Fax +1 250 472 4075. e-mail dlevin{at}uvic.ca
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Abstract |
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Introduction |
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Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) is a member of the Baculoviridae (Volkman et al., 1995 ) and is classified as a Group II NPV (Zanotto et al., 1993
; Bulach et al., 1999
). We previously sequenced the SpliNPV dnapol gene and demonstrated that the SpliNPV DNAPOL shares significant amino acid sequence similarity with family DNA polymerases and that the exonuclease domains and DNA polymerase motifs are highly conserved (Huang & Levin, 2001
). Alignment of NPV DNAPOL amino acid sequences revealed conserved amino acid motifs similar to those found in mammalian DNA polymerases (Huang & Levin, 2001
), which have been demonstrated to interact with a processivity factor, proliferating cell nuclear antigen [PCNA (Zhang et al., 1995
)].
One of our interests is directed toward structurefunction aspects that are unique to SpliNPV DNAPOL. Are the conserved amino acid residues in the N terminus of SpliNPV DNAPOL functionally important? Does purified SpliNPV DNAPOL interact specifically with cis-acting sequence elements within the SpliNPV non-hr origin of DNA replication? In this report, we have expressed the SpliNPV dnapol gene product using both prokaryotic and eukaryotic expression systems. We have characterized the functional activities of the full-length DNAPOL and a mutant DNAPOL, in which the first 80 amino acid residues were deleted. Using the SpliNPV non-hr origin of replication and a set of serial deletion clones as templates, we also demonstrated that SpliNPV DNAPOL can stimulate origin-dependent DNA replication in a cell-free system.
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Methods |
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PCR and cloning of the SpliNPV dnapol gene.
Based on DNA sequence analysis of SpliNPV dnapol (Huang & Levin, 2001 ), we designed two primers that amplify the full-length SpliNPV dnapol (Table 1
). In order to amplify a deletion mutation without the first 80 amino acids of the full-length DNAPOL, we employed an additional 39-mer forward primer (Table 1
). These primers were designed to create an insert with a BamHI and a NotI site on the amino and carboxyl termini of the DNA polymerase gene, respectively. All amplifications were carried out using Pfu DNA polymerase following the manufacturers instructions (Stratagene). The amplification products (3·1 kb for full-length and 2·8 kb for the
80 mutant) were gel purified, digested with BamHI and NotI, and ligated into vectors pGEX-5X-1 (Pharmacia) or pFastBacHTc (baculovirus expression system; Life Technologies) following the manufacturers instructions.
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All procedures were carried out at 4 °C unless otherwise specified. The bacterial cell pellets were thawed and resuspended in 10 vols (mass to volume) of lysis buffer (20 mM TrisHCl pH 8·0, 250 mM NaCl, 0·5 mM EDTA, 0·1% Triton X-100, 60 µg/ml lysozyme, 0·2 mM PMSF and 0·7 µg/ml leupeptin). The lysate was sonicated on ice until it lost viscosity and then centrifuged for 1 h at 4000 g in a Beckman SS34 rotor. The pellet and supernatant were analysed via 8% SDSPAGE. Fusion proteins were purified by affinity chromatography using glutathioneSepharose 4B contained in GST Purification Modules (Pharmacia). Cleavage of the desired proteins from GST was achieved using a site-specific protease, Factor Xa. Fusion proteins were detected using an immunoassay provided in the GST Detection Module, following the manufacturers protocol.
Expression and purification of SpliNPV DNAPOL in Sf9 cells.
The full-length SpliNPV dnapol and the 80 mutant gene products were hyper-expressed in Sf9 cells from a recombinant AcMNPV using the Bac-to-Bac Expression System (Life Technologies). Two recombinant plasmids, pFastBacHT-DNAPOL and pFastBacHT-
80DNAPOL, were constructed by subcloning the full-length dnapol and the
80 mutant PCR products into the BamHI and NotI sites of pFastBacHTc. Recombinant bacmids and AcMNPV were subsequently prepared according to the manufacturers instructions. Sf9 cells were infected at an m.o.i. of 10 and cells were harvested at various time points between 24 and 72 h post-infection (p.i.). The recombinant proteins, designated HisDNAPOL and His-del80DNAPOL, respectively, were purified on NiNTA agarose from Sf9 cells infected with recombinant baculovirus. Expression and purification of the fusion proteins were monitored by SDSPAGE analysis (Laemmli, 1970
). The purified polymerases were stored at -80 °C in buffer containing 65% glycerol.
SDSPAGE and Western blot analysis.
Protein concentrations were determined by the Bradford method (Bradford, 1976 ) with BSA as a standard. For Western blotting, proteins were electrophoresed by SDSPAGE and transferred to Immobilon-NC membranes (Millipore). The membranes were blocked with 5% nonfat milk and probed with a 1:5000 dilution of primary rabbit anti-His polyclonal antibodies. Membranes were then incubated with a 1:2000 dilution of goat anti-rabbit IgGhorseradish peroxidase conjugates, and developed using Enhanced Chemiluminescence (Amersham) substrates.
DNA templates.
Singly primed single-stranded M13mp18 was made by annealing 25 pmol of M13 universal forward primer (5' TGTAAAACGACGGCCAGT 3') to 2·5 pmol of single-stranded M13 DNA. The mixtures, containing 50 mM TrisHCl (pH 7·5), 5 mM MgCl2 and 100 mM NaCl in 50 µl, were heated to 100 °C and slowly cooled to room temperature. To prepare template for exonuclease assays, reaction mixtures (50 µl) containing 50 mM TrisHCl, pH 7·5, 1 mM dithiothreitol, 5 mM MgCl2, 0·4 mM each of dCTP, dGTP and dTTP, 20 µCi [-32P]dATP (3000 Ci/mmol), 500 µg/ml activated calf thymus DNA and 10 units of Klenow enzyme were incubated at 37 °C for 30 min, quenched by addition of 2·5 µl 0·5 M EDTA, and chilled on ice. DNA substrate was extracted twice with phenolchloroformisoamyl alcohol and purified by gel filtration using TE buffer.
DNA polymerase assay.
Reaction mixtures contained 50 mM TrisHCl, pH 8·0, 50 µg BSA, 0·5 mM dithiothreitol, 15 mM MgCl2, 200 mM KCl, 0·1 mM each of dCTP, dGTP and dTTP, 0·0125 mM dATP, 0·5 µCi [-32P]dATP (3000 Ci/mmol), 25 µg activated calf thymus DNA (Sigma) and purified DNA polymerase in a reaction volume of 100 µl. After incubation at 37 °C for 30 min, the reactions were stopped by addition of 100 µl 25 mM EDTA, 25 mM sodium pyrophosphate and 50 µg/ml of salmon sperm DNA followed by 1 ml 10% trichloroacetic acid (TCA). After 10 min on ice, the mixture was filtered through GF/C glass filters. The filters were washed twice with 2 ml 1 M HCl, 0·05 M sodium pyrophosphate, rinsed with ethanol, dried and counted in a liquid scintillation counter. One unit of DNA polymerase activity was defined as the amount of enzyme required to incorporate 1 nmol [
-32P]dNTP into acid-insoluble material/min at 37 °C.
DNA polymerase activity was also evaluated in the presence of various concentrations of MgCl2, KCl and (NH4)2SO4. The effect of aphidicolin was examined by addition of various concentrations of this DNA polymerase inhibitor (Pedrali-Noy & Spadari, 1980 ; Sheaff et al., 1991
). When inhibition of DNA polymerase activity by aphidicolin was measured, the concentration of dCTP was lowered to 10 µM. Thermostability of the DNA polymerase activity was examined by incubation of the SpliNPV DNAPOL at 50 °C for various lengths of time prior to its addition to the reaction mixture. All reactions were repeated in triplicate. Statistical analyses (ANOVA) were calculated using Microsoft Excel 97.
For DNA synthesis on singly primed, single-stranded circular M13 template, reaction mixtures (50 µl) contained 20 fmol substrate DNA, 20 mM Trisacetate (pH 7·3), 75 mM potassium acetate, 5 mM magnesium acetate, 1 mM DTT, 0·5 mM ATP, 60 µM each dGTP, dATP and dTTP, 20 µM [-32P]dCTP (3000 Ci/mmol), 50 µg/ml BSA and 200 fmol purified DNA polymerase. The reaction was incubated at 37 °C and terminated by addition of an equal volume of stop buffer (1% SDS, 40 mM EDTA, 60 µg/ml sonicated calf thymus DNA). The reaction products were precipitated with ethanol, resuspended in 20 µl sample buffer (0·1 M NaOH, 5% glycerol, 1 mM EDTA, 0·025% bromocresol green) and separated on a 1% alkaline agarose gel as described (Sambrook et al., 1989
). Dried gels were exposed and visualized by autoradiography.
3'5' exonuclease assay.
Exonucleolytic activities were determined in the absence of dNTPs under the conditions of the DNA polymerase assay. Reaction mixtures (100 µl) were incubated for 30 min at 37 °C with 25 µg activated calf thymus DNA containing 0·15 µg (6·7x105 c.p.m./µg) of 32P-labelled exonuclease substrate. Reactions were terminated by chilling on ice and by adding 20 µl 0·25 M EDTA, pH 8·0, 5 mg/ml BSA, and 20 µl 100% TCA. After centrifugation (13000 g, 30 min, 4 °C) the radioactivity of supernatant fractions (100 µl) was determined in a liquid scintillation counter. One unit of 3'5' exonuclease activity was defined as the amount of enzyme required to release 1 pmol [-32P]dCTP into acid-soluble material in 30 min at 37 °C.
The 3'5' exonuclease activity was also evaluated in the presence of various concentrations of MgCl2, KCl, (NH4)2SO4 and aphidicolin. The thermostability of the exonuclease activity was examined as outlined above. All reactions were carried out in triplicate. Statistical analyses were carried out as above.
Plasmid construction and transient replication assays.
Identification and DNA sequence analysis of the SpliNPV non-hr origin of DNA replication were reported previously (Huang & Levin, 1999 ). To identify the minimal origin sequence, we constructed a set of unidirectional nested deletions of the origin sequence using the Exo Mung Bean Deletion Kit (Clontech). Transient replication assays were performed as described (Huang & Levin, 1999
). Briefly, plasmid DNAs were transfected into 5x106 Sf9 cells by calcium phosphate precipitation. After 24 h at 27 °C, cells were infected with SpliNPV. Cells were harvested and DNA was extracted following the protocol of Sarisky & Hayward (1996)
. To test for replication in cells, DNA was digested with HindIII to linearize the plasmid, and with DpnI, which cleaves in the sequence 5'-CAGT-3' but only if the A is methylated. Plasmids that replicate in Sf9 cells due to the presence of a viral origin of DNA replication no longer possess the E. coli pattern of methylation at the DpnI site, and are therefore resistant to DpnI cleavage.
After electrophoresis on 0·7% agarose gels, the DNA was transferred to nylon membranes (Hybond-N, Amersham) and hybridized with [32P]dCTP-labelled pBlueScriptII DNA. Replicated plasmid DNAs are detected on Southern blots as HindIII linearized molecules. Plasmid DNAs that do not replicate retain the E. coli pattern of DpnI methylation and are detected on Southern blots as a ladder of fragments. All transient replication assays were repeated at least three times.
Preparation of cell extracts and cell-free replication.
Nuclear extracts from SpliNPV-infected Sf9 cells were prepared as reported previously (Huang & Levin, 1999 ). Total protein concentrations were determined by the Bradford method. The extracts were frozen in liquid nitrogen and stored at -80 °C. The cell-free replication assay was adapted from methods described by Stillman & Gluzman (1985)
. Briefly, a 25 µl reaction mixture contained 40 ng DNA templates and 100 µg of nuclear protein extract prepared from SpliNPV-infected Sf9 cells in 20 mM HEPES buffer, pH 7·5, 200 µM each UTP, GTP and CTP, 4 mM ATP, 100 µM each dATP, dGTP and dTTP, 25 µM dCTP, 40 mM phosphocreatine and 100 µg/ml creatine phosphokinase (Sigma). Purified SpliNPV DNAPOL (250 ng) was added to one set of reactions, while a second set was conducted in the absence of purified SpliNPV DNAPOL. The reaction mixtures were preincubated for 1 h at 37 °C, and then 2·5 µCi of [
-32P]dCTP (3000 Ci/mmol) was added to the reactions. Incubation was continued for another hour at 37 °C. Reactions were terminated by adding 200 µl 20 mM TrisHCl, pH 7·5, 10 mM EDTA, 0·1% SDS and 20 µg/ml RNase A, followed by incubation at 37 °C for 15 min. Protease K was added to 200 µg/ml and incubation was continued for another 30 min at 37 °C. The reaction mixtures were extracted with phenol and chloroformisoamyl alcohol (24:1), precipitated with 2·5 M ammonium acetate, 95% ethanol, and analysed by 0·8% agarose gel electrophoresis. Gels were dried, exposed, and quantified using a PhosphorImager (Molecular Dynamics).
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Results |
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DNA polymerase activity
Comparison studies revealed that the specific activities of proteins prepared using the two different systems were indistinguishable (Table 2). The DNAPOLs were most active at pH 7·5 (data not shown). The enzyme required moderate concentrations of divalent cations for activity. Maximum DNA polymerase activity was observed at MgCl2 concentrations of 1020 mM, while a concentration of 100 mM MgCl2 was inhibitory (Fig. 2A
). No activity was detected in the absence of divalent cations. Maximum SpliNPV DNAPOL polymerase activity was observed at KCl concentrations of 100200 mM. The polymerase activity was greatly reduced at a KCl concentration of 300 mM and was inhibited at higher salt concentrations (Fig. 2B
). Only 20% of residual activity could be detected in the presence of 400 mM KCl. While the SpliNPV DNAPOL was active at 100 mM (NH4)2SO4, only 40% activity could be detected in the presence of 300 mM (NH4)2SO4 (Fig. 2C
). Higher concentrations of ammonium ions strongly inhibited the enzymes (data not shown). The differences in DNA polymerase activity observed with 10 mM and 20 mM MgCl2, as well as with 100 mM and 200 mM KCl, were not statistically significant.
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3'5' exonuclease activity
The 3'5' exonuclease activities of the full-length DNAPOL and the 80 mutants from the two expression systems were high. The 3'5' exonuclease activity required Mg2+ ions (Fig. 3A
). Peak exonuclease activity was observed at MgCl2 concentrations of 1020 mM (Fig. 3A
) and at KCl concentrations of 100200 mM (Fig. 3B
). The differences in exonuclease activity observed with 10 and 20 mM MgCl2, as well as with 100 and 200 mM KCl, were not statistically significant. The inclusion of 200 mM (NH4)2SO4 reduced exonuclease activity of all four enzymes by 30% (Fig. 3C
).
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Replication of singly primed single-stranded M13 DNA
Having observed that the DNAPOL utilized gapped, activated calf thymus DNA as primer-templates, we further investigated the ability of DNAPOL to replicate single-stranded DNA. The18-mer oligonucleotide M13 DNA primer was incubated with single-stranded M13 DNA, purified DNA polymerase from the baculovirus expression system, and three deoxyribonucleoside triphosphates. Radiolabelled M13 DNA products were detected after 3 min of incubation. A strong signal was detected by 5 min after addition of dCTP, indicating that the SpliNPV DNAPOL synthesized DNA molecules of approximately 7200 nucleotides, corresponding to the entire length of M13 DNA (Fig. 4). The singly primed replication assay using single-stranded M13 DNA revealed that the
80 mutant was also capable of synthesizing a 7200 nucleotide replication product (data not shown). These results demonstrated that the DNA synthesis activity of SpliNPV DNA polymerase is processive.
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Replication of the non-hr origin of SpliNPV with DNAPOL and nuclear extracts
Using the SpliNPV non-hr origin fragment (E4) and the set of origin deletion clones, we established a cell-free DNA replication system for SpliNPV. Origin-dependent DNA replication was observed with nuclear extracts prepared from SpliNPV-infected Sf9 cells plus purified SpliNPV DNAPOL expressed from recombinant AcMNPV in Sf9 cells (Fig. 5C). Replication products were strongest with the E4 fragment containing the full-length SpliNPV non-hr origin. As observed with in the transient replication assays, the plasmid containing the
23 fragment supported minimal replication in the presence of nuclear proteins and the strength of the replication product signal increased as the length of the origin fragment increased. Plasmids containing the
14 and
15 fragments supported replication better than the plasmid containing the
23 fragment, but the strength of the replication products from these plasmids was less than that generated when the plasmid containing the E4 fragment was used as template. No replication signals were detected when plasmids either containing the
33 fragment or without origin-specific elements (pBlueScriptII) were used as template. A very low level of origin-dependent DNA replication was observed with nuclear extracts prepared from SpliNPV-infected Sf9 cells in the absence of purified SpliNPV DNAPOL (Fig. 5D
).
These experiments suggest that origin-dependent DNA replication occurred in both the transient replication assays and in cell-free replication assays using nuclear extracts prepared from SpliNPV-infected cells. Nuclear protein extracts prepared from SpliNPV-infected Sf9 cells supported a low level of origin-dependent DNA replication. The addition of purified SpliNPV DNAPOL to the reaction mix dramatically increased the strength of the replication product signal detected. Moreover, the strength of this origin-dependent replication signal was a function of specific DNA sequence elements within the origin fragments. Deletion of the 22 bp palindromic sequence (P2) overlapping the putative NFI transcription factor binding site abolished in vitro replication, and the strength of the replication products increased dramatically when the A+T-rich/NFIII sequences were included in the cloned fragments.
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Discussion |
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Our observations that the N-terminal truncated form of SpliNPV DNAPOL (80DNApol) possesses the same biochemical characteristics in vitro as full-length DNAPOL appear to be consistent with a study of mammalian DNAPol
(Schumacher et al., 2000
). When DNA synthesis was measured using a purified DNAPol
, the full-length and
NPol
(missing the first 80 amino acids) were equally active, suggesting that the 80 amino acids at the N terminus do not function in catalysis per se (Schumacher et al., 2000
). Further functional analyses, however, indicated that the holoenzyme containing
NPol
was significantly less efficient and slower than that containing full-length Pol
, suggesting that the N-terminal part of Pol
is involved in interactions with other proteins, such as SSB and RPC (Schumacher et al., 2000
). While the mammalian Pol
is about 100 amino acids longer than the SpliNPV DNAPOL, the first 80 amino acid residues of the two sequences aligned when subject to Clustal W analysis (data not shown). Further investigation is needed to address the importance of the first 80 amino acid residues of the SpliNPV DNAPOL during viral DNA replication in vivo.
Two types of cis-acting elements which function as viral origins of DNA replication have been identified in NPVs (hr and non-hr elements) by use of transient replication assays in which plasmid DNAs replicate in the presence of virus in NPV-infected insect cell lines (Kool et al., 1995 ). Non-hr origins of DNA replication (non-hr oris) have been described from AcMNPV (Kool et al., 1994
), Orgyia pseudotsugata MNPV (Pearson et al., 1993
), Spodoptera exigua MNPV (Heldens et al., 1997
) and Spodoptera littoralis NPV (Huang & Levin, 1999
). The non-hr ori of AcMNPV (the HindIII-K fragment) is enriched in defective AcMNPV genomes (Lee & Krell, 1994
) and appears to function as an origin of DNA replication during AcMNPV infection in vivo (Habib & Hasnain, 2000
). Thus, there is evidence that suggests that non-hr oris do play an important role in NPV replication, but the molecular mechanisms of this function are not known.
Non-hr oris have sequence elements that are similar to those found in origins of DNA replication of many eukaryotic DNA viruses (Kool et al., 1994 , 1995
; DePamphilis, 1996
). These comprise both a core element, which often consists of direct repeats and/or imperfect palindrome sequences, flanked by an A+T-rich region, and one or more auxiliary components that are composed of transcription-factor binding sites and/or promoter elements. The core element is absolutely required for replication. The presence of the auxiliary elements can stimulate the initiation of replication, but are not essential for it and are dispensable (DePamphilis, 1996
). In order for viral DNA synthesis to begin, sequence-specific recognition events, mediated by one or more initiator proteins (origin-binding proteins) encoded by the virus, are usually required (DePamphilis, 1996
). Following the initiation events, the origin-binding proteins recruit other replication proteins to the initiation site to unwind DNA, to synthesize new DNA primers and to elongate the synthesized DNA from both strands.
We conducted both transient replications assays and in vitro replication assays with purified SpliNPV DNAPOL using a set of deletion clones of the E4 fragment containing the putative SpliNPV non-hr ori as template. The results of these experiments suggest that a putative origin core element is located in a region between the two HinfI sites on the E4 fragment. This region contains a 22 bp imperfect palindrome sequence (5' CGGtGATCTGGCCAGATCgCCG 3'), which overlaps a putative NFI transcription-factor binding site. Deletion of this region abolished both plasmid replication in the transient replication assays and ori-dependent replication in the in vitro replication assays with purified SpliNPV DNAPOL. Based on these results, we hypothesize that the 22 bp imperfect palindrome sequence and/or the putative NFI binding site constitute the core element of the SpliNPV non-hr ori and that this sequence represents an origin-binding protein site.
Our analyses indicated that the A+T-rich sequence upstream of the 22 bp imperfect palindrome sequence also plays an important role in origin activity. Deletion of the A+T-rich sequence dramatically decreased replication in both the transient and in vitro replication assays. The region at the 5' end of the E4 fragment contains many repeat sequences and putative transcription-factor binding sites. Deletion of this region had little effect on replication in either the transient or the in vitro replication assays, and it may represent an auxiliary component that is not essential for origin activity.
In conclusion, our data suggest that the SpliNPV E4 fragment contains sequence elements that can support plasmid replication in transient replication assays and that purified SpliNPV DNAPOL can utilize these elements to synthesize DNA in vitro, in a sequence-dependent manner. While our data do not demonstrate conclusively that the SpliNPV E4 fragment functions as an origin of DNA replication during virus replication in vivo, they do suggest that there are specific interactions between SpliNPV DNAPOL and sequences within the putative SpliNPV non-hr element.
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Acknowledgments |
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References |
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Received 2 November 2000;
accepted 13 March 2001.