Initiation of DNA Replication at Palindromic Telomeres Is Mediated by a Duplex-to-Hairpin Transition Induced by the Minute Virus of Mice Nonstructural Protein NS1
1998; Elsevier BV; Volume: 273; Issue: 2 Linguagem: Inglês
10.1074/jbc.273.2.1165
ISSN1083-351X
AutoresKurt Willwand, Eleni Mumtsidu, Gaëlle Simon, Jean Rommelaere,
Tópico(s)Viral Infections and Outbreaks Research
ResumoThe 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 ATPγS (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. 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 ATPγS (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. 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 (1Kornberg A. Kornberg A. Baker T.A. DNA Replication. W. H. Freeman and Co., New York1992: 275-306Google Scholar), priming through a DNA-bound protein in the case of adenovirus, some bacteriophages and various linear plasmids (2Salas M. Miller J.T. Leis J. DePamphilis M.L. DePamphilis M.L. DNA Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 131-176Google Scholar), as well as self-priming at hairpins created by the folding-back of terminal palindromic sequences (3Cavalier-Smith T. Nature. 1974; 250: 467-470Crossref PubMed Scopus (175) Google Scholar). Palindromic termini are present in poxvirus (4Geshelin P. Berns K.I. J. Mol. Biol. 1974; 88: 785-796Crossref PubMed Scopus (122) Google Scholar, 5Baroudy B.M. Venkatesan S. Moss B. Cell. 1982; 28: 315-324Abstract Full Text PDF PubMed Scopus (194) Google Scholar) and parvovirus (6Bourguignon G.J. Tattersall P.J. Ward D.C. J. Virol. 1976; 20: 290-306Crossref PubMed Google Scholar, 7Hauswirth W.W. Berns K.I. Virology. 1979; 93: 57-68Crossref PubMed Scopus (47) Google Scholar) telomeres, paramecium mitochondrial DNA (8Pritchard A.E. Cummings D.J. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7341-7345Crossref PubMed Scopus (33) Google Scholar), and tetrahymena rDNA (9Karrer K.M. Gall J.G. J. Mol. Biol. 1976; 104: 421-453Crossref PubMed Scopus (157) Google Scholar). 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 (10Moyer R.W. Graves R.L. Cell. 1981; 27: 391-401Abstract Full Text PDF PubMed Scopus (86) Google Scholar, 11Moss B. Winters E. Jones E.V. Cozarelli N.R. Mechanism of DNA Replication and Recombination. Alan R. Liss, Inc., New York1983: 449-461Google Scholar, 12Berns K.I. Fields B.N. Knipe D.M. Howley P. Fundamental Virology. Raven Press, Ltd., New York1996: 2173-2197Google Scholar, 13Cotmore S.F. Tattersall P. Semin. Virol. 1995; 6: 271-281Crossref Scopus (72) Google Scholar, 14Baldauf A.Q. Willwand K. Mumtsidu E. Nüesch J. Rommelaere J. J. Virol. 1997; 71: 971-980Crossref PubMed Google Scholar, 15Willwand K. Baldauf A.Q. Deleu L. Mumtsidu E. Costello E. Beard P. Rommelaere J. J. Gen. Virol. 1997; 78: 2647-2655Crossref PubMed Scopus (17) Google Scholar). Mutational analyses indicated the existence of specific sequence elements within the core of poxvirus DNA palindromes, which are required for the formation of hairpins (16DeLange A.M. McFadden G. J. Virol. 1987; 61: 1957-1963Crossref PubMed Google Scholar). 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), 1The 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; ATPγS, adenosine 5′-O-(thiotriphosphate). a prototype member of the autonomously replicatingparvoviridae (17Siegl G. Bates R.C. Berns K.I. Carter B.J. Kelly D.C. Kurstak E. Tattersall P. Intervirology. 1985; 23: 61-73Crossref PubMed Scopus (226) Google Scholar), makes use of a hairpin-priming mechanism to replicate its linear, 5149-nucleotide (nt) (18Astell C.R. Gardiner E.M. Tattersall P. J. Virol. 1986; 57: 656-669Crossref PubMed Google Scholar) 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 (19Tattersall P. Ward D.C. Nature. 1976; 263: 106-109Crossref PubMed Scopus (144) Google Scholar, 20Cotmore S.F. Tattersall P. Adv. Virus Res. 1987; 33: 91-173Crossref PubMed Scopus (396) Google Scholar). 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) (14Baldauf A.Q. Willwand K. Mumtsidu E. Nüesch J. Rommelaere J. J. Virol. 1997; 71: 971-980Crossref PubMed Google Scholar). Such closed forms were also detected in parvovirus-infected cells (21Cotmore S.F. Gunther M. Tattersall P. J. Virol. 1989; 63: 1002-1006Crossref PubMed Google Scholar, 22Löchelt M. Delius H. Kaaden O.-R. J. Gen. Virol. 1989; 70: 1105-1116Crossref PubMed Scopus (9) Google Scholar). Further processing of cRF DNA in vitro requires the MVM nonstructural protein NS1 (14Baldauf A.Q. Willwand K. Mumtsidu E. Nüesch J. Rommelaere J. J. Virol. 1997; 71: 971-980Crossref PubMed Google Scholar, 15Willwand K. Baldauf A.Q. Deleu L. Mumtsidu E. Costello E. Beard P. Rommelaere J. J. Gen. Virol. 1997; 78: 2647-2655Crossref PubMed Scopus (17) Google Scholar). 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 (15Willwand K. Baldauf A.Q. Deleu L. Mumtsidu E. Costello E. Beard P. Rommelaere J. J. Gen. Virol. 1997; 78: 2647-2655Crossref PubMed Scopus (17) Google Scholar). 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 (14Baldauf A.Q. Willwand K. Mumtsidu E. Nüesch J. Rommelaere J. J. Virol. 1997; 71: 971-980Crossref PubMed Google Scholar, 23Tattersall P. Crawford L.V. Shatkin A.J. J Virol. 1973; 12: 1446-1456Crossref PubMed Google Scholar). Restoration of hairpin structures at the duplex right-hand telomere of MVM dsDNA templates (14Baldauf A.Q. Willwand K. Mumtsidu E. Nüesch J. Rommelaere J. J. Virol. 1997; 71: 971-980Crossref PubMed Google Scholar, 15Willwand K. Baldauf A.Q. Deleu L. Mumtsidu E. Costello E. Beard P. Rommelaere J. J. Gen. Virol. 1997; 78: 2647-2655Crossref PubMed Scopus (17) Google Scholar, 24Cossons N. Faust E.A. Zannis-Hadjopoulos M. Virology. 1996; 216: 258-264Crossref PubMed Scopus (15) Google Scholar, 25Cossons N. Zannis-Hadjopoulos M. Tam P. Astell C.R. Faust E.A. Virology. 1996; 224: 320-325Crossref PubMed Scopus (12) Google Scholar) and formation of dimer-length RF DNA (14Baldauf A.Q. Willwand K. Mumtsidu E. Nüesch J. Rommelaere J. J. Virol. 1997; 71: 971-980Crossref PubMed Google Scholar) 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 (26Fried M. Feo S. Heard E. Biochim. Biophys. Acta. 1991; 1090: 143-155Crossref PubMed Scopus (46) Google Scholar, 27Stillman B. Annu. Rev. Cell. Biol. 1989; 5: 197-245Crossref PubMed Scopus (285) Google Scholar, 28Clayton D.A. Annu. Rev. Cell. Biol. 1991; 7: 453-478Crossref PubMed Scopus (532) Google Scholar, 29McMurray C.T. Wilson W.D. Douglass J.O. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 666-670Crossref PubMed Scopus (48) Google Scholar, 30Spiro C. Richards J.P. Chandrasekaran S. Brennan R.G. McMurray C.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4606-4610Crossref PubMed Scopus (47) Google Scholar, 31Rasmussen C. Leisy D.J. Ho P.S. Rohrmann G.F. Virology. 1996; 224: 235-245Crossref PubMed Scopus (22) Google Scholar). The cultivation of A9 cells in suspension and the preparation of cytosolic extracts were carried out as described previously (14Baldauf A.Q. Willwand K. Mumtsidu E. Nüesch J. Rommelaere J. J. Virol. 1997; 71: 971-980Crossref PubMed Google Scholar). Nuclear extracts were prepared according to the method of Dignam (32Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9159) Google Scholar). Cultivation of Sf9 insect cells, infection with recombinant baculovirus expressing either wild type NS1 or the NS1 mutant K405R (33Nüesch J.P. Cotmore S.F. Tattersall P. Virology. 1995; 209: 122-135Crossref PubMed Scopus (79) Google Scholar), extract preparation, and NS1 purification by affinity chromatography were performed as reported (14Baldauf A.Q. Willwand K. Mumtsidu E. Nüesch J. Rommelaere J. J. Virol. 1997; 71: 971-980Crossref PubMed Google Scholar). The NS1 mutant K405R producing recombinant baculovirus was generously provided by Jesper Christensen (The Royal and Agricultural University of Copenhagen, Frederiksberg, Denmark). The MVM-specific insert (nt 1–5068) of the original MVM p98 plasmid (34Antonietti J.-P. Sahli R. Beard P. Hirt B. J. Virol. 1988; 62: 552-557Crossref PubMed Google Scholar) was recloned into the pGEM5Zf vector (Promega) (35Willwand K. Hirt B. J. Virol. 1993; 67: 5660-5663Crossref PubMed Google Scholar). Purified plasmid DNA was digested with SalI restriction enzyme and used as template DNA in the in vitroDNA replication assay. In vitro DNA replication was carried out as described previously (14Baldauf A.Q. Willwand K. Mumtsidu E. Nüesch J. Rommelaere J. J. Virol. 1997; 71: 971-980Crossref PubMed Google Scholar, 15Willwand K. Baldauf A.Q. Deleu L. Mumtsidu E. Costello E. Beard P. Rommelaere J. J. Gen. Virol. 1997; 78: 2647-2655Crossref PubMed Scopus (17) Google Scholar). Standard reaction mixtures (50 μl) consisted of 40 mm HEPES-KOH (pH 7.6), 8 mmMgCl2, 0.5 mm dithiothreitol, 100 μm each dATP, dGTP, and dTTP, 5 μCi of [α-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 theEscherichia coli DNA polymerase I (0.03 units). In some experiments, ATPγS was added in indicated amounts. The reaction was started by the addition of 200 ng (unless specified differently) ofSalI-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 were performed using the Mulfold computer program (36Jaeger J.A. Turner D.H. Zuker M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7706-7710Crossref PubMed Scopus (781) Google Scholar, 37Zuker M. Science. 1989; 244: 48-52Crossref PubMed Scopus (1726) Google Scholar, 38Jaeger J.A. Turnerand D.H. Zuker M. Methods Enzymol. 1990; 183: 281-306Crossref PubMed Scopus (376) Google Scholar). 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 byK = exp ΔG/RT. Proteins were separated by discontinuous SDS-polyacrylamide gel electrophoresis (39Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207192) Google Scholar) 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 (40Cotmore S.F. Tattersall P. J. Virol. 1986; 58: 724-732Crossref PubMed Google Scholar) at a 1:1000 dilution. Protein-bound antibodies were detected with peroxidase-conjugated specific antibodies and revealed by using the ECL system (Amersham). 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. 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 (34Antonietti J.-P. Sahli R. Beard P. Hirt B. J. Virol. 1988; 62: 552-557Crossref PubMed Google Scholar). 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.2 A (lane 1), doublet bands were detected around the anticipated positions ofPshAI 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. 2 B). The species forming the upper doublet bands in Fig. 2 A 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 ofSalI-digested p98 DNA (Fig. 2 B, lanes 1 and 3). In contrast, the species forming the lower doublet bands in Fig. 2 A were much more retarded under denaturing conditions (Fig. 2 B, lanes 2 and4). This is in line with the assumption that the lower doublet bands represent in vitro replication products with covalently closed termini (hairpin forms, h).Figure 2Hairpin-primed MVM DNA replication in extract of uninfected A9 cells. A, SalI-digested p98 DNA was incubated with 20 μg of cytosolic proteins plus increasing amounts (5, 10, or 20 μg) of nuclear proteins (lanes 1–3, respectively), or with increasing amounts (25, 30, or 35 μg) of cytosolic proteins alone (lanes 4–6, respectively). Product DNA was digested with PshAI and analyzed on a 5% polyacrylamide gel. B, individual DNA species obtained as inpanel A were recovered by electroelution and further analyzed on a 6% polyacrylamide/urea gel. HpaII-digested pGEM5Zf DNA was used as molecular weight marker. C, schematic representation of hairpin-primed DNA replication, including primer generation by a structural transition at the terminal palindromes and subsequent extension (solid lines). d, duplex; h, hairpin; v, viral strand;c, complementary strand; small arrowheadsindicate DNA strand 3′ ends.View Large Image Figure ViewerDownload Hi-res image Download (PPT) As described recently for a small fraction of in vitroprocessed MVM DNA (14Baldauf A.Q. Willwand K. Mumtsidu E. Nüesch J. Rommelaere J. J. Virol. 1997; 71: 971-980Crossref PubMed Google Scholar), the extended RF right-hand telomere undergoes a conformational transition from the duplex into the hairpin form, providing a primer for the reinitiation of DNA synthesis. According to Fig. 2 (A and B), this conformational transition appears to take place at both the MVM left and right-hand telomeres ofSalI-digested p98 DNA, followed by extension of the primers created in this manner. Therefore, together with our recent findings (14Baldauf A.Q. Willwand K. Mumtsidu E. Nüesch J. Rommelaere J. J. Virol. 1997; 71: 971-980Crossref PubMed Google Scholar, 15Willwand K. Baldauf A.Q. Deleu L. Mumtsidu E. Costello E. Beard P. Rommelaere J. J. Gen. Virol. 1997; 78: 2647-2655Crossref PubMed Scopus (17) Google Scholar) and the data of others (24Cossons N. Faust E.A. Zannis-Hadjopoulos M. Virology. 1996; 216: 258-264Crossref PubMed Scopus (15) Google Scholar, 25Cossons N. Zannis-Hadjopoulos M. Tam P. Astell C.R. Faust E.A. Virology. 1996; 224: 320-325Crossref PubMed Scopus (12) Google Scholar), the above results demonstrate hairpin-primed initiation of DNA replication at both the left- and right-hand inverted repeats of double-stranded MVM DNA, as depicted in Fig. 2 C. To gather information about the cellular factors involved in this terminal duplex-to-hairpin transition and primer extension, the initiation of DNA replication at SalI-digested p98 termini was further analyzed in the presence of varying amounts of cytosolic and nuclear extracts (Fig. 2 A). Increasing the portion of nuclear extract within the reaction mixture was marked by a dose-dependent enhancement of radioactive precursor incorporation into the component identified as the left-terminal hairpin form (band h, lanes 1–3) and by a concomitant decrease of the labeling of the left-terminal p98 duplex fragments (band d, lanes 1–3). Similarly, a stimulation of right terminal hairpin formation and extension was induced by nuclear extract components (compare lanes 1 and4) although no dose-dependent increase was observed (lanes 1–3). In the presence of cytoplasmic extract alone, left-end initiation was hardly detectable, and only a slight stimulation of right-end initiation was noted (lanes 4–6). 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 (14Baldauf A.Q. Willwand K. Mumtsidu E. Nüesch J. Rommelaere J. J. Virol. 1997; 71: 971-980Crossref PubMed Google Scholar). 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.3 A, 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 and2) or cytosolic plus nuclear (lanes 3 and4) 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. As shown in our previous reports (14Baldauf A.Q. Willwand K. Mumtsidu E. Nüesch J. Rommelaere J. J. Virol. 1997; 71: 971-980Crossref PubMed Google Scholar, 15Willwand K. Baldauf A.Q. Deleu L. Mumtsidu E. Costello E. Beard P. Rommelaere J. J. Gen. Virol. 1997; 78: 2647-2655Crossref PubMed Scopus (17) Google Scholar), NS1 is able to induce secondary rounds of in vitro nicking and extension of the right-hand telomere of MVM RF DNA in vitro, resulting in the release of the right-terminal hairpin DNA in a free form (depicted in Fig. 3 C, left-hand part; species H). This segregated hairpin species migrated in polyacrylamide gels at a similar position as the right-end turnaround MVM RF PshAI fragment (14Baldauf A.Q. Willwand K. Mumtsidu E. Nüesch J. Rommelaere J. J. Virol. 1997; 71: 971-980Crossref PubMed Google Scholar, 15Willwand K. Baldauf A.Q. Deleu L. Mumtsidu E. Costello E. Beard P. Rommelaere J. J. Gen. Virol. 1997; 78: 2647-2655Crossref PubMed Scopus (17) Google Scholar). It was thus necessary to ascertain that the in vitro labeled species h, derived from the right end of p98 MVM DNA, was the telomeric fragment of an RF molecule that underwent a conformational transition from duplex to hairpin (Fig. 3 C,right-hand part), rather than a free hairpin displaced from the NS1 nick site. To this end, p98 replication products obtained in the presence of NS1 were digested in parallel with the restriction enzymes PshAI, XbaI, or SspI, cleaving MVM DNA at different positions close to the right terminus (see Fig.1). As apparent from Fig. 3 B, the restriction digestion generated products migrating at the anticipated positions of right-handXbaI (706 bp), SspI (423 bp), andPshAI (132 bp) turnaround fragments. The size dependence of the low molecular weight DNA species on the restriction enzyme used (rather than on NS1 nicking at a unique telomeric site) indicates that the fast migrating h band from the PshAI-digested sample (Fig. 3 A, lanes 2 and 4) consists, at least for its major part, of the right-hand turnaround terminus of RF DNA, with little contribution of displaced free hairpins. In addition to h, another fragment, designated E, is visible in Fig. 3 A (lane 4). The apparent size of species E is in line with the assumption that it represents an extended right-hand PshAI fragment of full-length MVM DNA, in keeping with its comigration with this fragment in a neutral polyacrylamide gel (data not shown). Since p98 harbors a truncated palindrome at its right end, the question arises how this palindrome can be repaired to generate a full-length telomere. As schematized in Fig. 3 C, this can be assumed to result from right-end hairpin refolding and copying, followed by NS1-induced nicking and extension. 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 (33Nüesch J.P. Cotmore S.F. Tattersall P. Virology. 1995; 209: 122-135Crossref PubMed Scopus (79) Google Scholar), deficient for ATPase and helicase function, completely abolished hairpin-primed DNA replication (data not shown), confirming the requirement for NS1 in this reaction. NS1 may trigger replication from the duplex ends of MVM RF DNA by stimulating terminal hairpin refolding, elongation of the hairpin primer, or both. Should NS1 promote terminal hairpin formation, it was reasoned that the viral protein may induce the initiation ofSalI-digested p98 DNA replication in a defined reconstituted system supplemented with a purified DNA polymerase. In a first step, it was ascertained that without cell extract, no labeling of the DNA substrate took place in the presence of NS1 alone (data not shown), indicating that the NS1 preparation used was free of detectable DNA polymerase activity. Furthermore, the initiation of MVM dsDNA replication (as revealed by the nonappearance of labeled h bands) was not achieved with the Klenow fragment of E. coli DNA polymerase I in the absence of NS1 (Fig. 4 C, lane 1), confirming the inability of the Klenow enzyme to trigger the duplex-to-hairpin transition assumed to generate a primer-template structure. The labeling of duplex PshAI terminal fragments occurring under these conditions presumably resulted from the Klenow-mediated filling of substrate DNA recessed ends. In contrast, when the DNA substrate was incubated with both NS1 and Klenow polymerase, the turnaround telomeric fragments (h bands) became labeled (Fig. 4 C, lane 2), pointing to the formation and further extension of terminal hairpins. This was verified by performing control experiments similar to those shown in Figs. 2 B and3 B for the cell extract-mediated reaction, confirming that the newly synthesized DNA species visible in Fig. 4 C(lane 2) are indeed hairpin forms arising from structural transitions of the MVM RF duplex telomeres (data not shown). Equilibrium constants of 2.7 × 10−64m−1 and 2.2 × 10−14m−1 can be calculated for the duplex-to-hairpin conformational transitions at the left and truncated right end of p98 MVM DNA, respectively (36Jaeger J.A. Turner D.H. Zuker M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7706-7710Crossref PubMed Scopus (781) Google Scholar, 37Zuker M. Science. 1989; 244: 48-52Crossref PubMed Scopus (1726) Google Scholar, 38Jaeger J.A. Turnerand D.H. Zuker M. Methods Enzymol. 1990; 183: 281-306Crossref PubMed Scopus (376) Google Scholar). This makes it most unlikely that denaturation and refolding of the telomeres took place spontaneously, and NS1 only facilitated Klenow-driven elongation of the hairpin primers generated in this way. Consistently, preincubation of the DNA substrate in replication buffer at 37 °C failed to increase the yield of hairpin-primed replication upon subsequent addition of NS1 and Klenow polymerase (data not shown). Altogether, these observations argue for a direct role of NS1 in inducing the rearrangement of MVM RF duplex telomeres into hairpin structures that provide a primer for strand-displacement synthesis. 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 ATPγS. This analogue is known to compete with ATP for the binding to helicases and kinases while not being hydrolyzed (41Eckstein F. Annu. Rev. Biochem. 1985; 54: 367-402Crossref PubMed Google Scholar), 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. 5 A, 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 ATPγS (lane 4) and completely abolished by a 3-fold
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