Deutsches Krebsforschungszentrum, Applied Tumour Virology Program, Abteilung F0100, INSERM U375, Postfach 101949, 69009 Heidelberg, Germany1
University of Heidelberg, Department of Internal Medicine, Bergheimer Str. 58, 69115 Heidelberg, Germany2
Author for correspondence: Kurt Willwand. Fax +49 6221 424962. e-mail E.Burkard{at}dkfz-heidelberg.de
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Abstract |
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Main text |
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Although the structural transition of the right-end palindrome from linear duplex to hairpin can be driven by cellular factors to a limited extent in vitro (Cossons et al., 1996a , b
; Willwand et al., 1998
), efficient processing requires the viral NS1 protein (Willwand et al., 1998
). We recently proposed a molecular scheme for the structural transition process, involving the interaction of NS1 with [ACCA]2 motifs in the right-end MVM DNA palindrome (Willwand et al., 1998
). [ACCA]2 motifs constitute specific NS1-binding elements (Cotmore et al., 1995
) and might permit access of NS1 to the viral DNA terminus, thereby facilitating the local unwinding of the two DNA strands and their refolding into hairpins. Binding of NS1 to [ACCA]2 motifs within the MVM right-end palindrome, including those depicted in Fig. 1(A)
, was reported recently (Cotmore et al., 2000
). As part of an investigation into the importance of these motifs for the NS1-induced structural change in the MVM right-end palindrome, their binding affinities for NS1 were assessed further. Site-specific binding of NS1 to DNA was monitored according to Cotmore et al. (1995)
. Synthetic dsDNA oligodeoxynucleotides, in which the central region of the right-end palindrome was present in its wild-type (wt) form (wtAC) or harboured mutated [ACCA]2 motifs (
AC2 or
AC1/2) (Fig. 1A
), were 5' end-labelled using the Klenow fragment of Escherichia coli polymerase I, [
32P]dCTP, gel-purified and used as binding substrates. The degree of end labelling was comparable for all three oligodeoxynucleotides, as measured by autoradiography after polyacrylamide gel electrophoresis (Fig. 1B
, lanes 79). To analyse NS1-binding, the DNA substrates were incubated with recombinant NS1 followed by the addition of NS1-specific antibodies and immunoprecipitation of bound DNA (Cotmore et al., 1995
). As apparent from Fig. 1(B)
, wtAC and
AC2 DNA were both efficiently immunoprecipitated in the presence of NS1 (Fig. 1B
, lanes 3 and 5) but not in its absence (Fig. 1B
, lanes 4 and 6). In contrast, only a small portion of the
AC1/2 mutant, devoid of [ACCA]2 motifs, was recovered in the presence of NS1 (Fig. 1B
, lane 1). Residual
AC1/2 precipitation is likely to result from the reported nonspecific DNA-binding capacity of NS1 (Cotmore et al., 1995
). Altogether, these results are thus consistent with the specific binding of NS1 to wtAC and
AC2 via the [ACCA]2 motif(s).
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As depicted in Fig. 2(A), the duplex-to-hairpin transition occurring at the MVM right-end palindrome has been assumed to be facilitated as a result of NS1-binding to [ACCA]2 motifs on both sides of the axis of symmetry of the extended duplex palindrome, followed by unwinding of the c-strand DNA, cruciformation, branch migration and hairpin building (Willwand et al., 1998
). This model predicts that elimination of [ACCA]2 motifs from the right-end palindrome should interfere with the generation of hairpin primers by preventing NS1 from specifically binding to the palindrome. To test this hypothesis, the mutant MVM 5' telomeric clones, p
AC2 and p
AC1/2, were produced from plasmid p98 (Antonietti et al., 1988
) by substituting
AC2 or
AC1/2 DNA for the corresponding wt 87 bp region within the right-end palindrome. Since the unrelated elements with which the [ACCA]2 motifs II and II' were replaced in the mutant clones were inverted repeats, the 5' telomere of these clones retained the potential of the wt constructs for forming hairpins, at least as far as DNA sequence was concerned. The structural transition of the extended 5' telomere was determined using the wt and mutant DNA templates described above, linearized by SalI cleavage, in conjunction with a reconstituted in vitro system (Willwand et al., 1998
). This system allows NS1-induced hairpin formation at linear duplex termini and elongation of the hairpin 3' end by the Klenow fragment of E. coli DNA polymerase I. While hairpin-primed DNA replication is undetectable in this system in the presence of Klenow polymerase alone, the addition of NS1 suffices for its induction (Willwand et al., 1998
). Analysis of the in vitro-generated DNA involves PshAI digestion, yielding short right-end DNA fragments that can be readily separated based on their electrophoretic characteristics into refolded and elongated (product DNA) or outstretched fragments (input DNA) (Baldauf et al., 1997
). As shown in Fig. 2(B
, lanes 1, 3 and 5), in vitro replication reactions carried out in the absence of NS1 gave rise to one labelled PshAI product in amounts that were similar for wt and mutant DNA templates. This DNA product, marked d, had been identified in previous studies as the duplex (open) DNA right-end terminus, presumably labelled through filling of the SalI-restricted end of the linearized template DNA. An additional band, migrating much faster and more heavily labelled, was detected when replication was carried out in conjunction with NS1 and Klenow polymerase (Fig. 2B
, lane 2). This species, marked h, was previously assigned to the processed terminal hairpin fragment (Baldauf et al., 1997
). Importantly, the formation of this turn-around species was greatly reduced when either p
AC2 or p
AC1/2 was used as template instead of the wt form in the replication assay (Fig. 2B
, lanes 4 and 6). Inhibition of the right-end structural transition due to the alteration of both outward [ACCA]2 motifs (
AC2) was not reinforced by further mutating the inward motif (
AC1/2). This leads us to conclude that the former two elements (II and II') constitute codeterminants of NS1-induced remodelling of the duplex right-end telomere, although we did not formally exclude that motif I may cooperate with motifs II and II' in driving this reaction. It should be stated that, in the presence of NS1, the d band was reduced to a similar extent, irrespective of whether replication was carried out with wt or mutant input DNA (Fig. 2B
, lanes 4 and 6). This failure of d DNA induction may be assigned to the fact that, at least in the first place, only a very small proportion of template DNA gets remodelled and replicated in vitro (Baldauf et al., 1997
) and, in the second place, NS1 is encumbered with unspecific DNA-binding and nuclease activities that are liable to interfere with the end-labelling reaction (Cotmore et al., 1995
; data not shown). The residual amount of hairpin-primed replication from the terminus of
AC1/2 template DNA is likely to be due to the nonspecific DNA binding and helicase activities of NS1 (Cotmore et al., 1995
; Wilson et al., 1991
). Altogether, these results argue for a role of the [ACCA]2 motifs, present on both sides of the palindromes right-end axis of symmetry, as regulatory cis-elements mediating the NS1-induced structural transition of the MVM right-end terminus.
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Unwinding of dsDNA by helicases is usually independent of the DNA nucleotide sequence, as was shown for NS1 (Wilson et al., 1991 ). However, some viral helicases, such as the large T antigen of simian virus 40 (SV40), display both unspecific helicase activity and specific unwinding activity, the latter of which relies on the binding of the large T antigen to SV40 origin DNA and serves as the initiation of SV40 DNA replication (Dean et al., 1987
). The present study shows that the MVM NS1 protein, in addition to its role as a classical unspecific helicase, is also able to unwind the MVM right-end palindrome in a sequence-specific manner, participating in this way in the initiation of MVM RF DNA replication.
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Acknowledgments |
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References |
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Received 9 July 2001;
accepted 26 February 2002.