The right-end telomere of replicative form (RF) DNA of the autonomous parvovirus minute virus of mice (MVM) consists of a sequence that is self-complementary except for a three nucleotide loop around the axis of symmetry and an interior bulge of three unpaired nucleotides on one strand (designated the right-end ' bubble '). This right-end inverted repeat can exist in the form of a folded-back strand (hairpin conformation) or in an extended form, base-paired to a copy strand (duplex conformation). We recently reported that the right-end telomere is processed in an A9 cell extract supplemented with the MVM nonstructural protein NS1. This processing is shown here to result from the NS1-dependent nicking of the complementary strand at a unique position 21 nt inboard of the folded-back genomic 5h end. DNA species terminating in duplex or hairpin con-

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 3Ј-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 (3Ј-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 (5Ј-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 5Ј 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 dimerlength 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 ...

We have developed an in vitro system that supports the replication of natural DNA templates of the autonomous parvovirus minute virus of mice (MVM). MVM virion DNA, a single-stranded molecule bracketed by short, terminal, self-complementary sequences, is converted into double-stranded replicative-form (RF) DNA when incubated in mouse A9 fibroblast extract. The 3 end of the newly synthesized complementary strand is ligated to the right-end hairpin of the virion strand, resulting in the formation of a covalently closed RF (cRF) molecule as the major conversion product. cRF DNA is not further replicated in A9 cell extract alone. On addition of purified MVM nonstructural protein NS1 expressed from recombinant baculoviruses or vaccinia viruses, cRF DNA is processed into a right-end (5 end of the virion strand) extended form (5eRF). This is indicative of NS1-dependent nicking of the right-end hairpin at a distinct position, followed by unfolding of the hairpin and copying of the terminal sequence. In contrast, no resolution of the left-end hairpin can be detected in the presence of NS1. In the course of the right-end nicking reaction, NS1 gets covalently attached to the right-end telomere of the DNA product, as shown by immunoprecipitation with NS1-specific antibodies. The 5eRF product is the target for additional rounds of NS1-induced nicking and displacement synthesis at the right end, arguing against the requirement of the hairpin structure for recognition of the DNA substrate by NS1. Further processing of the 5eRF template in vitro leads to the formation of dimeric RF (dRF) DNA in a left-to-left-end configuration, presumably as a result of copying of the whole molecule by displacement synthesis initiated at the right-end telomere. Formation of dRF DNA is highly stimulated by NS1. The experimental results presented in this report support various assumptions of current models of parvovirus DNA replication and provide new insights into the replication functions of the NS1 protein.

No abstract