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

Abstract

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 ...

Genome Packaging Sense Is Controlled by the Efficiency of the Nick Site in the Right-End Replication Origin of Parvoviruses Minute Virus of Mice and LuIII

“…Relative origin activity was determined by PI quantitation of 32 P incorporation into DNA species migrating in each lane throughout the entire gel region shown here. Lanes 7 to 11 contain ScaI-digested, 32 P-labeled products from samples equivalent to those shown in lanes 1 to 5, following immunoprecipitation with anti-NS1 antibodies.…”

“…These could potentially anneal to create a minimal stem-loop structure with the nick site exposed on one side of the loop, as commonly found in rolling-circle replication initiation sites. (B) Autoradiograph of a neutral agarose gel showing ScaI-digested, 32 P-labeled in vitro replication products generated from substrates containing minimal viral origin sequences of 136 bp from the LuIII genome (lane 1 to 2) and 134 bp from the Lu⌬2 genome (lane 3) and substrates carrying minimal origins plus additional UTR sequences (563 to 565 bp total, lanes 4 to 6 as indicated), generated in the presence (lanes 2 to 5) or absence (lane 1 and 6) of NS1. The arrowheads at the left indicate the positions of linearized forms of the input substrate (4084 and 3246 bp, respectively, for the minimal and UTR-containing constructs).…”

“…In these assays, 32 P-labeled, hairpinned LuIII and Lu⌬2 origin sequences were incubated with recombinant NS1 and HMG1 in the presence of ATP (14). NS1 and HMG1 concentrations were titrated against both substrates to make sure the assays were linear with respect to these components, and several time points were investigated without revealing any significant difference in the efficiency of nicking at the two origins (data not shown).…”

Molecular and Structural Basis of the Evolution of Parvovirus Tropism

“…The hairpin transfer in the cross-linked ends of the duplex molecules results in a flip/flop sequence inversion (flip is the reverse complementary sequence of flop) and the creation of new replication origins by a terminal resolution reaction (Snyder et al, 1990). For parvoviruses with unique ends, there is a bias toward right-end initiation (Willwand et al, 1998). In addition to nuclear components, NS-1, a pleiotropic viral phosphoprotein of about 80 kDa, is critical in this event, by virtue of its site-specific nickase activity (Nuesch et al, 1995), but also for the generation of dimer duplex intermediates.…”

Searching for Palindromic Sequences in Primary Structure of Proteins

“…For example the nuclear glucocorticoid receptor recognizes a palindromic fragment of human DNA [17,18]. Moreover palindromic sequences are present in telomeres and are necessary for initiation of DNA replication therein [19,20], All in all palindromic sequences of DNA were studied in depth and their role is quite well understood [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. On the other hand the presence of palindromic sequences in primary structure of proteins has not gained as much attention [21,22].…”