From the Department of Applied Tumor Virology, Abteilung 0610, Deutsches Krebsforschungszentrum and Formation INSERM U375, D-69009 Heidelberg, Germany
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ABSTRACT |
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The linear single-stranded DNA genome of the
minute virus of mice (MVM) is replicated via a double-stranded
replicative form (RF) intermediate. Amplification of this RF is
initiated by the folding-back of palindromic sequences serving as
primers for strand-displacement synthesis and formation of dimeric RF
DNA. Using an in vitro replication assay and a cloned MVM
DNA template, we observed hairpin-primed DNA replication at both MVM
DNA termini, with a bias toward right-end initiation. Initiation of DNA
replication is favored by nuclear components of A9 cell extract and
highly stimulated by the MVM nonstructural protein NS1. Hairpin-primed
DNA replication is also observed in the presence of NS1 and the Klenow
fragment of the Escherichia coli DNA polymerase I. Addition
of ATPS (adenosine 5
-O-(thiotriphosphate)) blocks the
initiation of DNA replication but not the extension of pre-existing
hairpin primers formed in the presence of NS1 only. The NS1-mediated
unwinding of the right-end palindrome may account for the recently
reported capacity of NS1 for driving dimer RF synthesis in
vitro.
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INTRODUCTION |
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DNA polymerases are unable to copy unprimed DNA templates. Various
mechanisms have therefore evolved in different biological systems to
provide the template strand to be replicated with a free 3-hydroxyl
end that can be extended. These mechanisms include RNA priming in the
case of pro- and eucaryotes as well as some viruses (1), priming
through a DNA-bound protein in the case of adenovirus, some
bacteriophages and various linear plasmids (2), as well as
self-priming at hairpins created by the folding-back of terminal
palindromic sequences (3). Palindromic termini are present in poxvirus
(4, 5) and parvovirus (6, 7) telomeres, paramecium mitochondrial DNA
(8), and tetrahymena rDNA (9). The terminal palindromes of pox- and
parvovirus genomes play an essential role in distinct steps of viral
DNA replication, including the priming of concatemeric intermediates
formation and their subsequent resolution into monomeric daughter
molecules (10-15). Mutational analyses indicated the existence of
specific sequence elements within the core of poxvirus DNA palindromes, which are required for the formation of hairpins (16). Given that the
transition of palindromic DNA from the duplex into the hairpin
configuration requires the overcome of a high energetic barrier,
factor(s) interacting with specific DNA elements may be necessary for
this structural transition.
Minute virus of mice (MVM),1
a prototype member of the autonomously replicating
parvoviridae (17), makes use of a hairpin-priming mechanism
to replicate its linear, 5149-nucleotide (nt) (18) single-stranded (ss)
DNA genome. MVM DNA replication starts with complementary strand
synthesis primed at the genomic left-hand (3-terminal) hairpin,
producing a double-stranded (ds) replicative form (RF) DNA (19, 20). As
demonstrated recently in vitro for the majority of processed
DNA molecules, complementary strand synthesis stops when reaching the
folded-back right-hand (5
-terminal) hairpin, and is followed by
ligation of the newly synthesized and parental strands. This results in
a molecule covalently closed at both ends (cRF) (14). Such closed forms
were also detected in parvovirus-infected cells (21, 22). Further
processing of cRF DNA in vitro requires the MVM
nonstructural protein NS1 (14, 15). When added as a purified
polypeptide expressed from baculovirus vectors, NS1 was found to nick
the MVM complementary strand 21 nt inboard of the folded-back genomic
5
terminus, followed by initiation of strand-displacement synthesis
and copying of the hairpin to yield a molecule that is extended at its
right end (15). Rearrangement of the copied right-hand palindrome into
hairpin structures (formation of the so-called rabbit-eared configuration) provides a primer for reinitiation of replication in a
strand-displacement manner leading to the formation of concatemeric molecules, in particular dimer-length RF DNA (14, 23).
Restoration of hairpin structures at the duplex right-hand telomere of MVM dsDNA templates (14, 15, 24, 25) and formation of dimer-length RF DNA (14) were recently achieved in vitro. Interestingly, dimer formation was found to be stimulated to a great extent by the NS1 protein. Since the mechanism of this stimulation remained elusive, the present study was undertaken to determine whether NS1 promoted hairpin refolding, extension of the hairpin primer, or both. The present data argue for a role of NS1 in the transition of the extended terminal palindrome into hairpin structures. In this respect, parvoviruses provide a model for the involvement of protein-DNA interactions in the structural transition of DNA known to take part in various biological mechanisms, in particular on the level of DNA replication and transcription (26-31).
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EXPERIMENTAL PROCEDURES |
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Preparation of Cell Extracts and Production of NS1 from Recombinant Baculoviruses-- The cultivation of A9 cells in suspension and the preparation of cytosolic extracts were carried out as described previously (14). Nuclear extracts were prepared according to the method of Dignam (32). Cultivation of Sf9 insect cells, infection with recombinant baculovirus expressing either wild type NS1 or the NS1 mutant K405R (33), extract preparation, and NS1 purification by affinity chromatography were performed as reported (14). The NS1 mutant K405R producing recombinant baculovirus was generously provided by Jesper Christensen (The Royal and Agricultural University of Copenhagen, Frederiksberg, Denmark).
DNA Templates-- The MVM-specific insert (nt 1-5068) of the original MVM p98 plasmid (34) was recloned into the pGEM5Zf vector (Promega) (35). Purified plasmid DNA was digested with SalI restriction enzyme and used as template DNA in the in vitro DNA replication assay.
In Vitro DNA Replication and Analysis of Product
DNA--
In vitro DNA replication was carried out as
described previously (14, 15). Standard reaction mixtures (50 µl)
consisted of 40 mM HEPES-KOH (pH 7.6), 8 mM
MgCl2, 0.5 mM dithiothreitol, 100 µM each dATP, dGTP, and dTTP, 5 µCi of
[-32P]dCTP (4 Ci/mmol), 100 µM each CTP,
GTP, and UTP, 3 mM ATP, 40 mM phosphocreatine,
20 µg/ml creatine phosphokinase, baculovirus-produced purified NS1
(200 ng), and either cytoplasmic plus nuclear extract (35 and 10 µg,
respectively, unless specified) or the Klenow fragment of the
Escherichia coli DNA polymerase I (0.03 units). In some experiments, ATP
S was added in indicated amounts. The reaction was
started by the addition of 200 ng (unless specified differently) of
SalI-digested p98 template DNA, and carried out for 90 min at 37 °C. Restriction-digested replication products were analyzed on
5% polyacrylamide gels using Tris borate-EDTA as running buffer. Gel-purified DNA products were recovered by electroelution and analyzed
under denaturing conditions on 6% polyacrylamide/urea gels using the
same running buffer as above.
Thermodynamic Calculations--
Thermodynamic calculations were
performed using the Mulfold computer program (36-38). Free energy
values were calculated for the hairpin and duplex forms of the entire
left terminal repeat (nucleotides 1-115) and the truncated right
terminal repeat (nucleotides 4028-5068) of MVM DNA, at 37 °C and
100 mM Na+. The equilibrium constant for the
transition: duplex = 2 hairpins was calculated by
K = exp G/RT.
Western Blotting-- Proteins were separated by discontinuous SDS-polyacrylamide gel electrophoresis (39) and transferred to nitrocellulose membranes using a semidry blotting system (Bio-Rad). Filters were incubated for 1 h at room temperature in blocking buffer (4% nonfat dry milk in phosphate-buffered saline) and then for 2 h with an antiserum raised against the C-terminal part of NS1 (40) at a 1:1000 dilution. Protein-bound antibodies were detected with peroxidase-conjugated specific antibodies and revealed by using the ECL system (Amersham).
Glycerol Gradient Centrifugation-- Affinity-purified NS1 (10 µg in a volume of 300 µl) was layered onto a 15-40% glycerol gradient (1.4 ml) in 10 mM Hepes-KOH, pH 7.5, 5 mM MgCl2, 0.1 mM EDTA, 50 mM NaCl, 1 mM dithiothreitol. After centrifugation in a Beckman 55 rotor at 50,000 rpm for 18 h at 4 °C, 100-µl fractions were collected by pipetting from the top of the tube.
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RESULTS |
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Formation of Terminally Closed DNA from Cloned MVM DNA in A9 Cell Extract-- The infectious clone p98 comprises the MVM DNA sequence extending from nt 1 to 5068, including the entire left-hand and more than half of the right-hand inverted repeat (34). Digestion of p98 DNA with the restriction enzyme SalI releases the MVM-specific insert flanked by SalI linker sequences (depicted in Fig. 1). SalI-digested p98 DNA was used as a template in an in vitro replication reaction containing a mixture of cytosolic and nuclear extracts from A9 murine fibroblasts. The reaction products were digested with the restriction enzyme PshAI at nt 670 and 4916 (see Fig. 1) and first analyzed on a native polyacrylamide gel. As apparent from Fig. 2A (lane 1), doublet bands were detected around the anticipated positions of PshAI left- and right-hand terminal fragments produced from the p98 MVM DNA insert. When the individual bands were gel-purified and further analyzed under denaturing conditions, structural differences became evident (Fig. 2B). The species forming the upper doublet bands in Fig. 2A gave rise to denaturation products expected from the duplex forms (d) of the left-hand (670 bp) and right-hand (152 bp) MVM PshAI fragments of SalI-digested p98 DNA (Fig. 2B, lanes 1 and 3). In contrast, the species forming the lower doublet bands in Fig. 2A were much more retarded under denaturing conditions (Fig. 2B, lanes 2 and 4). This is in line with the assumption that the lower doublet bands represent in vitro replication products with covalently closed termini (hairpin forms, h).
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Terminal Initiation of MVM DNA Replication Is Stimulated by NS1-- We recently reported that the formation of MVM dimer RF DNA, initiated at the right-hand telomere of a monomeric RF DNA template, is stimulated by the nonstructural protein NS1 in vitro (14). Therefore, we were interested to test whether NS1 had any influence on hairpin-primed DNA synthesis at the termini of SalI-digested p98 MVM DNA. As illustrated in Fig. 3A, supplementing replication reactions with NS1 led to a dramatic increase in the amount of nucleotide precursors incorporated into DNA products terminating in a turnaround configuration (designated h), whereas the extended duplex products d became hardly detectable. This effect was irrespective of whether the reaction was performed in cytosolic (lanes 1 and 2) or cytosolic plus nuclear (lanes 3 and 4) extract. Thus, the viral NS1 protein proved able to stimulate hairpin-primed DNA replication at both the left and right ends of MVM RF DNA supplied as terminally extended substrate.
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Stimulation of Duplex-to-Hairpin Transition in the Presence of NS1-- The NS1 protein used in these experiments was expressed from a baculovirus vector in Sf9 insect cells and purified by affinity chromatography. Although giving a single NS1 band in Coomassie Blue-stained polyacrylamide gels (data not shown), this preparation may contain minor amounts of contaminating Sf9 proteins. To confirm that the above-mentioned stimulation of replication initiation was indeed due to NS1 rather than to a contaminating cellular factor, the affinity-purified NS1 was subjected to a second purification step by centrifugation through a glycerol gradient. Individual fractions of this gradient were tested for their NS1 content and their ability to initiate DNA replication at the ends of SalI-digested p98 substrate in the presence of cytoplasmic A9 cell extract. Initiation, as revealed by the appearance of the faster migrating bands h in Fig. 4, panel B, was most efficient in the presence of fraction 8 coinciding with the peak of NS1 in the gradient (panel A). This argues for a major role of NS1 in the initiation reaction. It should also be stated that the distribution of NS1 in the gradient was symmetrical, but the extent of initiation was not. This raises the possibility that (a) cellular factor(s) may additionally stimulate the NS1-induced reaction. Initiation at the left end seemed to require a higher amount of NS1 than initiation at the right end (see below). Replacing wild type NS1 by the baculovirus-produced mutant K405R (33), deficient for ATPase and helicase function, completely abolished hairpin-primed DNA replication (data not shown), confirming the requirement for NS1 in this reaction.
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Terminal Hairpin Formation Is ATP-dependent--
The
structural transformation of MVM DNA termini is likely to consume
energy. Therefore, we were interested to determine whether hairpin-primed MVM DNA replication was ATP-dependent and
performed competition experiments with ATPS. This analogue is known
to compete with ATP for the binding to helicases and kinases while not
being hydrolyzed (41), acting as an inhibitor. As mentioned above,
incubation of SalI-digested p98 DNA with Klenow polymerase only resulted in the labeling of duplex PshAI terminal
fragments (Fig. 5A, lane
1). Supplementing the replication reaction with NS1 led to a
limited hairpin-primed initiation of replication in the absence of
added ATP, as revealed by the appearance of labeled h species
(lane 2). Addition of ATP highly increased the efficiency of
replication initiation at both termini (lane 3). Replication
was reduced by supplying an equimolar amount of ATP
S (lane
4) and completely abolished by a 3-fold excess of this analogue (lane 5). We therefore conclude that the initiation of
hairpin-primed DNA replication at the termini of MVM duplex DNA is an
ATP-consuming process. The low level of replication initiation observed
in the absence of added ATP (Fig. 5A, lane 2) may
be due to residual ATP bound to NS1. Indeed, ATP binding and processing
have been shown to occur as part of the helicase function of NS1
(42).
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DISCUSSION |
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Initiation of DNA Replication at the Termini of Cloned MVM
DNA--
Replication of the parvovirus ssDNA genome involves its
initial conversion into a dsRF by extension of the left-hand (3) palindrome folded-back into a terminal hairpin structure that serves as
primer for the synthesis of a complementary strand (14, 19, 20). The
newly synthesized strand is ligated to the right-terminal hairpin, the
processing of which is driven by the parvovirus protein NS1, as was
recently characterized in our laboratory by means of an in
vitro replication assay (14, 15). Besides mediating the
site-specific nicking of the right-end hairpin and its extension into a
duplex structure, NS1 was found to stimulate strand-displacement synthesis, which starts from the duplex right telomere and eventually gives rise to a dimer-length RF intermediate (14, 15). The present work
was undertaken to investigate the mechanism by which NS1 promotes DNA
synthesis initiated at a duplex telomere. To this end, we made use of
an MVM DNA clone (p98) from which the MVM part can be excised, to give
an RF molecule terminating in telomeres of full (left-hand) or partial
(right-hand) size. The results presented in this report show that DNA
replication is initiated at both duplex DNA ends in a process that
includes unwinding of viral and complementary strands, hairpin
refolding, and primer extension. This process is driven by nuclear
components and highly stimulated by NS1. It should be stated that,
although truncated, the right telomere of p98-derived MVM DNA has more
than half the length of the original palindromic sequence and therefore
is still able to fold back into a hairpin primer. Consistently, when
p98 DNA was replaced by a natural MVM RF template terminating in
full-size duplex telomeres on both sides, similar results were obtained (data not shown). The MVM insert of plasmid p98 is bracketed by SalI linker sequences. As shown in Fig. 1, the 3
-hydroxyl
terminus on the right-hand side of MVM DNA excised by SalI
digestion, corresponds to the last nucleotide of the viral sequence and
can thus base pair with its complement upon folding-back of the
hairpin. On the left-hand side, the free hydroxyl group is carried by
the first flanking nucleotide of the linker sequence, G, which is complementary to the C present immediately downstream from the terminal
palindrome on the MVM viral strand. Therefore, the presence of linkers
does not result in mismatches that would interfere with full pairing of
the free end of either terminal palindromic sequence and with its use
as a primer for strand elongation.
NS1-induced Duplex-to-Hairpin Transition--
The ability of NS1
to stimulate hairpin-primed strand-displacement synthesis may take
place at the level of hairpin formation, primer extension, or both. To
test the role of NS1 in hairpin folding, replication was analyzed in
the absence of cell nuclear factors, which may also contribute to this
process, by incubating the DNA substrate with NS1 and the Klenow
fragment of E. coli DNA polymerase I. Hairpin formation was
revealed through the known capacity of the polymerase for strand
displacement synthesis (43), leading to the extension of the hairpin
primer. Whereas the Klenow fragment was unable by itself to induce
hairpin formation, hairpin-primed strand elongation was observed when
p98 MVM DNA was incubated with both NS1 and the bacterial polymerase.
This result strongly argues for an inducing effect of NS1 on hairpin
formation, although it does not rule out that NS1 facilitates in
addition the strand-displacement synthesis reaction. The involvement of
NS1 in the remodeling of MVM telomeres is consistent with the fact that
the viral enzyme is endowed with an ATP-dependent helicase
function (42), and that hairpin-primed replication could be fully
inhibited by an excess of ATPS competitor. Furthermore,
preincubation of the DNA substrate with NS1 plus ATP alone allowed
subsequent hairpin-primed DNA replication in the presence of ATP
S
upon addition of DNA polymerase. Therefore, NS1 appears able to drive
an ATP-dependent event that takes place in the absence of
DNA synthesis and primes ensuing elongation, most likely consisting in
a duplex-to-hairpin transition at both palindromic termini of MVM DNA.
This function of NS1 may account for the recent finding that the viral
protein stimulates the in vitro formation of dimer-length
DNA from a monomeric MVM RF substrate terminating in a turnaround
(left-hand side) and a duplex (right-hand side) conformation (14).
Mechanism of Hairpin Folding-- Alternative mechanisms, designated "indirect" and "direct" slippage, have been proposed for the formation of hairpins from duplex palindromes (49). As illustrated in Fig. 6A, the direct process (numbered 1) consists in whole duplex unwinding followed by rapid intrastrand reannealing to form hairpins, whereas the indirect mechanism (numbered 2) involves the initial formation of a cruciform structure around the axis of symmetry of the palindrome, followed by branch migration and hairpin formation. Kinetic studies with short synthetic palindromes suggest that one or the other process may occur in different systems, depending on the palindrome size and nucleotide sequence as well as on the salt concentration and pH (50-52).
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Conclusion-- In general, significant energetic barriers restrain duplex palindromes from folding into hairpins (29, 50-52), as exemplified by the above mentioned equilibrium constants calculated for the MVM DNA termini. Studies with synthetic palindromes have shown that the transition energetic extensively varies with environmental conditions such as salt concentration and pH (29, 50-52). In cells, changes in local pH may be brought about by the interaction of the palindrome with electrically charged side chains of proteins (29), facilitating the structural transition. This is in keeping with the possibility that NS1 may induce the conformational changes at the MVM RF DNA ends through a direct interaction with the terminal palindromes.
The interaction of specific protein(s) with distinct DNA regions, referred to as origins of replication, is a prerequisite for the induction of DNA replication in many viral, procaryotic and eucaryotic systems (1, 27, 28, 58-60). As in the parvovirus model, several origins contain a palindromic core sequence, e.g. those of mitochondrial DNA from yeast to men (61). Origin recognizing proteins take part in local DNA unwinding or influence replication in other not yet defined ways (62-67). As mentioned above, NS1 may interact with [ACCA]2 motifs present in the center of the terminal palindromes (57). Mutational analyses should indicate whether these sides mediate the NS1-induced generation of hairpin primers, besides their known contribution to the NS1-dependent processing of bridge junctions within MVM multimeric RF intermediates (68, 69). The rerearrangement of inverted terminal repeats into hairpins, proven to be involved in pox- and parvovirus-DNA replication (10-13), has also been proposed as an essential step in the amplification of the rRNA gene of Tetrahymena thermophila (70) and during gene amplification in mammalian cells (for a review, see Ref. 26). Furthermore, palindromic sequences have been found in the regulatory regions of eucaryotic genes (29, 30). Theoretical (71) and experimental (29, 30, 72) data from these systems indicate a role of palindromes in determining biologically relevant DNA secondary structures. Proteins involved in the conformational transition of palindromes may thus be ubiquitously distributed. The limited rearrangement of MVM DNA left- and right-hand palindromes into hairpins, as induced by nuclear extract in the absence of NS1, argues for the presence of A9 cell factors with a remodeling capacity for MVM DNA telomeres. In this respect, parvovirus DNA constitutes an interesting probe to identify cellular factors and target DNA sequences that are likely to be involved in the control of eucaryotic DNA replication or gene expression. ![]() |
ACKNOWLEDGEMENTS |
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We thank Bernhard Hirt for helpful discussions and critical reading of the manuscript and Andreas Baldauf for useful discussions. We are indebted to Jesper Christensen for providing the recombinant baculovirus producing NS1 mutant K405.
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FOOTNOTES |
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* This work was supported in part by the Commission of the European Communities (Biomedicine and Health Research Program).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These authors contributed equally to this work.
§ To whom correspondence should be addressed: Deutsches Krebsforschungszentrum, Abt. 0610, Postfach 101949, D-69009 Heidelberg, Germany. Tel.: 49-6221-424969; Fax: 49-6221-424962; E-mail: k.willwand{at}dkfz-heidelberg.de.
¶ Recipient of an Alexander von Humboldt Foundation fellowship.
1
The abbreviations used are: MVM, minute virus of
mice; ss, single-stranded; ds, double-stranded; RF, replicative form;
nt, nucleotide(s); bp, base pair(s); d, duplex; h, hairpin;
ATPS, adenosine 5
-O-(thiotriphosphate).
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REFERENCES |
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