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Previous Article | Next Article 
Journal of Virology, June 2003, p. 6245-6254, Vol. 77, No. 11
0022-538X/03/$08.00+0 DOI: 10.1128/JVI.77.11.6245-6254.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Departments of Laboratory Medicine,1 Genetics, Yale University School of Medicine, New Haven, Connecticut 065102
Received 9 December 2002/ Accepted 12 March 2003
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ABSTRACT |
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-32P]TTP during the synthesis. The observed products suggest a resolution model that explains conservation of the hairpin sequence and in which a novel heterocruciform intermediate plays a crucial role. In vitro, NS1 initiates two replication pathways from OriLTC, the single active origin embedded in one arm of the dimer junction. NS1-mediated nicking liberates a base-paired 3' nucleotide to prime DNA synthesis and, in a reaction we call "read-through synthesis," forks established while the substrate is a linear duplex synthesize DNA in the flop orientation, leading to DNA amplification but not to junction resolution. Nicking leaves NS1 covalently attached to the 5' end of the DNA, where it can serve as a 3'-to-5' helicase, unwinding the NS1-associated strand. In the second pathway, resolution substrates are created when such unwinding induces the palindrome to reconfigure into a cruciform prior to fork assembly. New forks can then synthesize DNA in the flip orientation, copying one cruciform arm and creating a heterocruciform intermediate. Resolution proceeds via hairpin transfer in the extended arm of the heterocruciform, which releases one covalently closed duplex telomere and a partially single-stranded junction intermediate. We suggest that the latter intermediate is finally resolved via an NS1-induced single-strand nick at the otherwise inactive origin, OriLGAA. |
INTRODUCTION |
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MVM DNA is amplified through a series of monomeric and concatemeric duplex replicative-form (RF) intermediates by a unidirectional, leading-strand-specific, cellular replication fork, aided and orchestrated by a single multifunctional viral initiator protein, NS1 (13, 18). Like other RCR initiators, NS1 is a site-specific duplex DNA binding protein with site-specific, single-strand nickase activity. It activates replication by binding to its duplex recognition sequence, (ACCA)n, present in all viral origins, and introducing a strand-specific, single-strand nick at an adjacent initiation site. This transesterification reaction creates a base-paired 3' nucleotide to serve as a primer for new DNA synthesis while leaving NS1 covalently attached to the 5' end of the DNA at the nick site, where it likely serves as a 3'-to-5' helicase (9). The available evidence suggests that NS1 may also operate the toggle-switch mechanisms which unfold and refold the viral hairpins (22, 23), although these reactions remain to be elucidated in detail.
The initial rolling hairpin model (21) predicted that any terminus which was not a perfect palindrome would be produced with equal frequency in two sequence orientations, called flip and flop, which are the inverted complements of one another. While this prediction holds for the MVM right-end hairpin, the left (viral 3') end of the genome is present in both packaged virus and intracellular viral RF DNA in only a single orientation, dubbed flip, as shown in Fig. 1 (1, 2). Moreover, while the right end of the genome serves as an NS1-dependent origin in its hairpin configuration (3, 15), the origin associated with the left end is silent until the hairpin is extended and copied during replication to form the fully base-paired, dimer bridge region, which spans adjacent genomes in dimer RF (10, 17).
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PIF and NS1 bind site specifically to both oriLTC and oriLGAA, but these interactions are only cooperative on oriLTC, forming a ternary complex that effectively stabilizes the interaction of NS1 with OriLTC during ATP hydrolysis (6). Like other RCR initiators, NS1 can only nick single-stranded forms of its initiation site (19). Nicking at duplex oriLTC thus requires ATP hydrolysis to fuel the unwinding activity that melts the duplex substrate and presents the nick site to the nickase as single-stranded DNA. Without this cooperative interaction between NS1 and PIF, the duplex nick site remains silent, as in oriLGAA. This distinction between the ability of NS1 to nick single-stranded forms of its nick site unaided by cofactors and the strict requirement for cofactors to allow nicking at duplex forms is an important aspect of the resolution model presented here.
This tight control means that NS1 is only able to introduce one single-stranded nick into the duplex bridge sequence, into the active initiation site in the lower strand of the B arm, as shown in Fig. 1. A new fork initiated at this site on a linear substrate would synthesize DNA in the flop orientation, ultimately recreating the linear duplex and displacing one positive-sense single strand. We observed this type of reaction in the experiments described here and designate it "read-through synthesis." We suggest that it represents an amplification, rather than a full resolution, pathway and that such products are not the only outcome of initiation at OriLTC.
We have previously shown that templates containing the dimer bridge sequence as part of a supercoiled plasmid can be resolved in vitro using cellular replication extracts and recombinant NS1 to generate two new duplex viral termini, one associated with each arm of the junction palindrome. This resolution was found to be somewhat asymmetric, as shown in Fig. 1, generating predominantly NS1-associated "extended forms" of the A arm, while the B arm was most frequently represented as "turn-around" hairpin forms in which the two strands of the duplex were covalently continuous through the axis of symmetry (10, 17).
Here we reexamine the dimer resolution reaction in vitro, using linear substrates and develop a resolution model based on the structure and temporal expression of three resolution intermediates, which we call MJ1, MJ2, and 
J, and on the distribution of newly synthesized DNA. Evidence is presented that supports the characterization of MJ2 as a heterocruciform structure. The proposed mechanism relies on characteristic parvoviral duplex-to-hairpin rearrangements of the palindrome, mediated by NS1, to explain bridge resolution and the generation of negative-sense genomes with left-end telomeres exclusively in the flip orientation.
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MATERIALS AND METHODS |
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-32P]TTP and unlabeled dATP.
Resolution assays.
Recombinant histidine-tagged MVM NS1 was expressed in HeLa cells from recombinant vaccinia virus and purified by nickel-chelate chromatography as previously described (20). Recombinant RPA, expressed from baculovirus vectors and purified as described elsewhere (9), was kindly provided by Jesper Christensen. Replication extracts were prepared from uninfected A9 cells essentially as described by Wobbe et al. (24), except that they were dialyzed against 20 mM Tris-HCl (pH 7.5), 0.2 mM EDTA, 25 mM NaCl, 0.5 mM dithiothreitol, 10% glycerol, and 10% sucrose, which concentrated the extracts approximately twofold. Assays were carried out as previously described (11), in a volume of 20 µl that contained recombinant NS1 (2.5 µg/ml), deoxynucleotides, MgCl2, ribonucleotides, ATP, and an ATP-regenerating system, and either 32P-3'-end-labeled template DNA (1.25 µg/ml) or unlabeled, blunt-ended template DNA plus [-32P]TTP. Reactions were terminated by addition of sodium dodecyl sulfate (SDS) and EDTA to final concentrations of 0.5% and 5 mM, respectively, and analyzed on neutral agarose gels (one dimension) or neutral followed by alkaline agarose gels (two dimensions), as described previously (11). Samples to be immunoprecipitated were incubated in buffer containing 2% SDS at 60°C for 20 min to disrupt noncovalent interactions, diluted, and precipitated with rabbit antiserum directed against the N-terminal domain of NS1. Unless otherwise specified, neutral agarose gels contained 0.2% SDS in both gel and buffer.
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RESULTS |
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-32P]TTP into newly synthesized DNA.
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J) which are potential resolution intermediates. In the following analyses we show, in detail, the structure of each of these products and intermediates.
As observed previously using supercoiled dimer bridge substrates labeled by [-32P]TTP incorporation (10), resolution of the linear substrates appeared asymmetric, in that extended forms of the A arm greatly outnumbered its turn-around form. However, in the present analysis, extended and turn-around forms of the B arm often appeared approximately equimolar and were greatly outnumbered by duplex forms of the A arm.
The native agarose gel shown in Fig. 2B contained SDS, allowing fragments that were covalently associated with NS1 to enter the gel. When a sample equivalent to the one shown in lane 3 was digested with proteinase K prior to electrophoresis (lane 4), potential intermediates MJ1, MJ2, J, and extended forms of the A and B arms showed enhanced mobility, indicating that they were initially covalently complexed with NS1. In contrast, unit-length input molecules and turn-around forms of the A and B arms were not NS1 associated and remained in the unbound fraction following immunoprecipitation (Fig. 2C, lane 2).
Since MJ1 and MJ2 forms migrated more slowly than input DNA even after proteinase K digestion, they likely contained more DNA than the input, while J migrated faster than input, suggesting that it had lost DNA. None of the observed forms comigrated with denatured template (Fig. 2B, lane 8), confirming that they were not simply melted-out forms of the substrate.
(ii) Products labeled by incorporating [-32P]TTP into newly synthesized DNA.
In the absence of NS1, unlabeled linear fragments containing the dimer bridge were barely labeled in vitro (Fig. 2B, lane 5). Introducing NS1 into the reaction mixture (lane 6) resulted in the appearance of newly synthesized DNA in a variety of species, including unit-length linear molecules that comigrated with the 3'-end-labeled input DNA shown in lane 1. Digestion with proteinase K did not affect the mobility of this product (lane 7), and it did not immunoprecipitate with anti-NS1 serum (Fig. 2C, compare lanes 6 and 7), indicating that it was not covalently associated with NS1 even though NS1 was required for its synthesis (Fig. 2B, compare lane 5 with lanes 6 and 7). We suggest that these labeled unit-length molecules are the products of read-through replication (discussed below), which allows DNA amplification without junction resolution.
Surprisingly, the resolution product which contained most of the newly synthesized DNA was the turn-around form of the B arm. This product was identified as a B turn-around form because it migrated at 
0.5 kb under neutral conditions but migrated at 1 kb under denaturing conditions (see below), indicating that the strands of the duplex were covalently continuous at one end. In addition, this DNA species was not directly associated with NS1, as confirmed by its presence in the unbound fraction of the anti-NS1 immunoprecipitate shown in Fig. 2C, lane 6.
All fully resolved forms of the bridge (i.e., extended and turn-around forms of both the A and B arms) contained at least some newly synthesized DNA, as did the NS1-associated MJ1 and MJ2 intermediates (Fig. 2C, lane 7). In contrast, the J intermediate was not significantly labeled by in vitro synthesis (compare Fig. 2B, lane 4 with 7, and 2C, lane 3 with 7), suggesting that it did not contain significant stretches of newly synthesized DNA.
Read-through synthesis.
Unit-length duplex molecules labeled by incorporation of [-32P]TTP into newly synthesized DNA in the NS1-dependent reaction (Fig. 2B, lanes 6 and 7, and C, lanes 5 and 6) are products of a type of reaction we call read-through synthesis, which does not lead directly to the generation of duplex resolution products. In the amplification pathway depicted in Fig. 3A, a leading-strand-specific cellular fork, which can be assembled in vitro with recombinant RPA, RFC, PCNA, and DNA polymerase (S. F. Cotmore, J. Christensen, and P. Tattersall, unpublished results), forms rapidly on the newly available 3' hydroxyl (step i), allowing strand displacement synthesis to occur on the linear template (step ii). This displaces an NS1-associated, single-stranded, positive-sense form of the A arm, while replacing it with labeled, newly synthesized DNA (step iii). Displaced single-stranded forms of the A arm were identified in the resolution products of 3'-end-labeled substrates, but they migrated as a diffuse band in the SDS-agarose gels shown here and were minor products, perhaps because single-stranded DNA is more susceptible to degradation than duplex DNA in complex cell extracts. In Fig. 3B, lane 1, we show proteinase K-digested products of an NS1-driven reaction displayed on a non-SDS gel. Single-stranded forms of the A arm can be identified in these products, but they become particularly apparent when compared to products shown in lane 2, where the release of single-stranded forms has been promoted by the inclusion of a large molar excess of recombinant RPA. Their identity as NS1-associated 1.6-kb single strands was confirmed by two-dimensional gel electrophoresis and by their specific immunoprecipitation with anti-NS1 serum (data not shown).
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Time course of the resolution reaction.
Products generated and labeled by incorporation of [-32P]TTP into newly synthesized DNA were analyzed, at intervals during the course of a 2-h reaction, by electrophoresis through neutral SDS-agarose gels, both before (Fig. 4A, lanes 2 to 8) and after (Fig. 4A, lanes 9 to 12) proteinase K digestion. During the first 90 min of the reaction, the rate of [-32P]TTP incorporation into DNA was approximately linear, but declined thereafter. The clear redistribution of incorporated label with time, from higher-molecular-weight structures at the earliest time points to the final resolution products after 1 to 2 h, suggests that this reaction was relatively synchronous and that initiation was most common early during synthesis, perhaps because active NS1 concentrations became limiting thereafter.
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Generation and processing of 32P-3'-end-labeled substrates were also analyzed as a function of time in an NS1-dependent reaction (Fig. 4B). Analysis of total reaction products harvested at 30-min intervals (lanes 1 to 3) and species immunoprecipitated from reactions with anti-NS1 serum (lanes 4 to 6) showed that the heterogeneous MJ1 species appeared early and declined with time, while the J intermediate progressively accumulated. Residual input DNA remained in the unbound fraction of these precipitates (lanes 7 to 9), together with turn-around forms of the resolution products.
Structures of MJ1, MJ2, and J intermediates.
Products generated and labeled by incorporation of [-32P]TTP into newly synthesized DNA from the 60-min time point were digested with proteinase K (as in Fig. 4A, lane 10) and further analyzed by two-dimensional electrophoresis under native and then denaturing conditions (Fig. 5A). This type of analysis allowed us to distinguish extended forms of the telomeres, where the two strands of the duplex were free to separate under denaturing conditions, from turn-around forms, where the two strands were held together covalently at one end. Thus, extended forms of the B arm migrated as 0.5-kb duplexes in the first dimension and as 0.5-kb single strands in the second, while turn-around forms of the B arm migrated as somewhat smaller duplexes than extended forms but appeared almost twice as long (approximately 1 kb) when denatured.
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2-kb duplexes in the first dimension and as 2-kb single strands in the second, indicating that the new DNA was contained within full-length DNA strands. In contrast, none of the new DNA in the MJ1 and MJ2 intermediates was associated with a full-length strand. Instead, most of the new DNA in the MJ2 intermediate was present in a single strand of approximately 0.5 kb which comigrated in the alkaline dimension with extended forms of the B arm. In Fig. 5D we suggest a heterocruciform structure for this MJ2 intermediate, in which new DNA is confined to an extended form of the B arm. This newly synthesized DNA is predicted to be in the flip orientation, but this has not been formally confirmed. New DNA associated with the more heterogeneous MJ1 intermediate migrated as an extended diagonal in the alkaline dimension, ranging from the position of an extended form of the B arm to the size of a B turn-around form in the more slowly migrating duplex MJ1 forms (Fig. 5A). Extrapolating from the MJ2 structure depicted in Fig. 5D, we suggest that MJ2 forms are likely converted to MJ1 forms by a hairpin transfer event that melts out the strands of the lower, extended-form, duplex and allows them to fold back on themselves, repositioning the 3' nucleotide so that it primes synthesis back along the B arm. Accordingly, MJ1 forms would be heterogeneous, because they include a spectrum of molecules in which the returning fork has progressed to different lengths back along the B arm.
Two-dimensional analysis of 3'-end-labeled products (Fig. 5B) indicated that MJ1 and MJ2 intermediates were relatively minor species. Although the 3'-end-labeled forms associated with these intermediates were faint, they clearly contained one strand of 1.6 kb, corresponding to a 3'-end-labeled form of the lower strand that had been nicked in the B arm at OriLTC, and one full-length strand of 2.1 kb. This full-length strand is difficult to see for the MJ2 intermediate in Fig. 5B, since it migrates coincidentally with the residual input substrate; however, it can be readily detected in two-dimensional gels of 3'-labeled products immunoprecipitated by virtue of their covalent association with NS1, as shown at higher magnification in Fig. 5C, since residual input is not present in these precipitates. Thus, MJ2 (and MJ1) and products are in accord with those detailed in the proposed heterocruciform structure of the MJ2 intermediate as shown for 3'-end-labeled substrates in Fig. 5E, having one 3'-labeled intact strand and one 3'-labeled strand that has been nicked at OriLTC, creating an extended form of the A arm.
Most notably, labeled forms of the B arm (0.5 to 1 kb) were not apparent in the 3'-end-labeled MJ intermediates shown in Fig. 5B, indicating that the B forms labeled so heavily by in vitro synthesis in Fig. 5A were most likely produced by nicking and extension of the lower strand of the B arm, which would not carry a 3' end label, as illustrated in Fig. 5E.
Figures 5B and C also reveal the structure of the J intermediate. This structure was not visible in Fig. 5A because it contained no newly synthesized DNA, but in the products of the 3'-end-labeled substrates it could be seen to contain an intact 2.1-kb strand and a 1.6-kb extended-form A arm. Since it is also present in Fig. 5C it must carry a covalently associated NS1 molecule, but it migrates faster than substrate DNA in the neutral dimension, so that it must have lost some DNA, presumably the unlabeled lower strand of the B arm. We therefore suggest that J molecules (together with free turn-around forms of the B arm) are the products of MJ1 intermediates in which the returning fork has completed synthesis of the B turn-around structure, causing it to be released.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In Fig. 6 we suggest a heterocruciform resolution model to explain how NS1 is able to bring about resolution of the dimer bridge into separate duplex telomeres while conserving the flip configuration of the hairpin on all negative-sense strands. This model takes into account the structures and apparent kinetic relationships between the various resolution intermediates and products observed above.
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Formation of the cruciform resolution substrate. In order for resolution to occur, we suggest that the initial nicking event in OriLTC must be followed by melting and rearrangement of the bridge palindrome into a cruciform intermediate prior to fork assembly, allowing synthesis of new DNA in the flip orientation. Such duplex-to-hairpin rearrangements could be accomplished by a simple helicase mechanism, as illustrated in Fig. 6, steps ii to iv. In this pathway assembly of the fork is delayed, so that in the presence of RPA the NS1 3'-to-5' helicase activity begins to unwind the duplex, as recently documented (9). As the helicase passes through the axis of symmetry, the two separated strands begin to fold back on themselves, and branch migration continues until the cruciform arms extend past the nick site (steps iii and iv).
Alternatively, NS1 may be able to melt out and reconfigure the MVM dimer bridge palindrome in an RPA-independent reaction. Willwand and colleagues (23) observed NS1-dependent, hairpin-primed replication at both termini of a duplex MVM DNA template using cell extracts in vitro, and they went on to show that purified NS1 was able to melt out extended forms of the MVM right-end telomere in the absence of cellular replication factors (22). This latter reaction was dependent upon the presence of both copies of the paired NS1 binding sites which surround the symmetry axis of the right-end telomere. Although plausible, at present there is no direct experimental evidence for comparable reactions involving extended-form left-end telomeres of MVM or the dimer bridge.
Synthesis of the heterocruciform. Once the cruciform extends to include sequences beyond the nick site in the A arm, the primer exposed at the nick site in OriLTC can undergo a template switch by annealing with its complement in the lower cruciform arm (Fig. 6, step v). If a replication fork assembles after this point, the resulting synthesis will unfold and copy the flop sequence of the lower cruciform arm, with NS1 perhaps again serving as the 3'-to-5' helicase required for strand displacement. This would create a heterocruciform intermediate that contains a newly synthesized telomere in the flip orientation attached to the lower (-) strand of the B arm (step vi). Such a structure corresponds exactly to that of the MJ2 intermediate, as determined in Fig. 5.
We had previously suggested that a cellular or viral recombinase could resolve heterocruciform intermediates of this type, with directional specificity, into the expected termini (13, 14). However, such a reaction would transfer the labeled, newly synthesized DNA (depicted in step vi) onto an extended form of the A arm, leaving the B turn-around arm unlabeled. Since this is not compatible with our experimental data, in which turn-around forms of the B arm are seen to accumulate most of the newly synthesized DNA, we now conclude that a directional recombinase is not responsible for subsequent resolution.
Hairpin transfer and release of the B turn-around arm. We suggest that synthesis pauses after completion of the MJ2 intermediate because the fork reaches the end of its template strand (Fig. 6, step vi). Although evidence of a precursor-product relationship has not been established for the subsequent transition, we suggest that MJ2 forms are converted to MJ1 forms by a hairpin transfer event that allows the two strands of the lower heterocruciform arm to melt out and fold back on themselves, repositioning the exposed 3' nucleotide on the B arm (Fig. 6, step vii) and allowing a new fork established on this primer to synthesize a B turn-around form (step viii). This hairpin transfer reaction could be mediated by either of the NS1-driven mechanisms discussed previously in relation to dimer bridge substrates. Thus, according to the new model, both creation of the heterocruciform and its subsequent resolution would be driven by the characteristic NS1-mediated duplex-to-hairpin rearrangements of the palindrome that are the hallmark of parvovirus DNA amplification.
Upon completion of the B turn-around structure, it would be released from the MJ1 intermediate (Fig. 6, step ix), leaving a J intermediate which consists of one intact DNA strand base-paired to an extended form of the A arm. Since the A arm of this intermediate has an NS1-associated, recessed 5' end, the 3'-to-5' helicase activity of NS1 would again tend to unwind the lower strand of the palindrome, allowing the single strands to fold back on themselves and creating the branched intermediate depicted in Fig. 6.
Introduction of a single-strand nick and resolution of the J intermediate.
In the absence of a large excess of RPA, the helicase activity of NS1 rarely manages to fully displace the lower strand of the J intermediate (Fig. 3B), but it likely creates a dynamic structure in which the nick site in the inactive A arm (OriLGAA) is transiently, but repeatedly, exposed in a single-stranded form. We speculate that under these conditions OriLGAA can be nicked by free NS1 in a PIF-independent reaction (Fig. 7, steps i and ii). This type of single-stranded nicking reaction could also occur on MJ1 and MJ2 intermediates, perhaps bypassing release of the J form. However, J forms do accumulate, suggesting that the postulated single-strand nicking reaction may be inefficient or that NS1 becomes limiting at late times in vitro.
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Although all other termini generated in this resolution pathway are in the flip orientation, the final single-stranded nick would generate a positive-sense strand with a flop hairpin in which the three-nucleotide bubble sequence, GAA, is positioned in the outer arm. Although such telomeres were not observed in vivo for MVM (1), they have been identified in the closely related virus LuIII (5). Unlike MVM, LuIII packages DNA strands of both senses with equal efficiency (4), and while the left-end hairpins of its negative-sense strands are all in the flip orientation, in positive-sense strands flip and flop orientations are expressed with equal frequency (5). We suggest that there could be two different mechanisms for processing J intermediates and that the balance between these that is achieved in vivo is determined by the rate of hairpin transfer at the right end of the viral genome. Since the orientation of termini excised by these two mechanisms is somewhat different, one way to test this prediction experimentally would be to impair the efficiency of the MVM right-end origin by genetic manipulation.
Preliminary experiments indicate that nature has already explored this avenue. We are currently mapping the sequences in the LuIII genome which allow it to package all of the resolution products predicted from the heterocruciform model, and we find that this ability maps to a 2-nucleotide insertion in the NS1 nick site of the right-end origin. This insertion has the effect of making the LuIII origin refractory to nicking when compared in vitro to its MVM counterpart. Thus, when the right-end hairpin of LuIII is substituted for the equivalent MVM sequence, a virus is generated which has a slow right-end origin and which packages, in addition to negative-sense strands, positive strands whose left-end termini exist in both flip and flop orientations (S. F. Cotmore and P. Tattersall, unpublished data).
This observation suggests that any genome-length single strand which is excised from replicative-form concatemers will ultimately be packaged, and that it is only the failure to excise certain termini efficiently which precludes their encapsidation. Hence, it predicts that there are no specific sequences in MVM or LuIII DNA that allow the selection of negative- versus positive-sense strands for packaging. In this respect the heterocruciform model potentially provides a mechanistic perspective from which to view the earlier kinetic hairpin transfer model of parvovirus DNA replication (5). This mathematical model was based purely on physical measurements of terminal sequence heterogeneity and the ratio of plus to minus strands packaged into virions by different parvoviruses, and it suggested that all such heterogeneity could be explained simply by the relative rates of hairpin transfer at the right and left ends of each genome. Although this model had many interesting implications, it was difficult to reconcile with our knowledge of viral biochemistry, since we have long been aware that left-end termini of viruses such as MVM and LuIII are excised by resolution of the dimer intermediate and not simply by hairpin transfer reactions. The data presented in this paper may therefore provide a conceptual basis for reconciling the earlier mathematical model with specific biochemical mechanisms.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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