Abstract
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 ATPgammaS (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.
Highlights
DNA polymerases are unable to copy unprimed DNA templates
When added as a purified polypeptide expressed from baculovirus vectors, NS1 was found to nick the Minute virus of mice (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]
Terminal Initiation of MVM DNA Replication Is Stimulated by NS1—We recently reported that the formation of MVM dimer replicative form (RF) DNA, initiated at the right-hand telomere of a monomeric RF DNA template, is stimulated by the nonstructural protein NS1 in vitro [14]
Summary
DNA polymerases are unable to copy unprimed DNA templates. Various mechanisms have evolved in different biological systems to provide the template strand to be replicated with a free 3Ј-hydroxyl end that can be extended. 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]. 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 proteinDNA 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 (26 –31)
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