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.
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.
Willwand
K
K
Department of Applied Tumor Virology, Abteilung 0610, Deutsches Krebsforschungszentrum and Formation INSERM U375, D-69009 Heidelberg, Germany. k.willwand@dkfz-heidelberg.de
Mumtsidu
E
E
Kuntz-Simon
G
G
Rommelaere
J
J
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004261:DNA Replication; D004279:DNA, Viral / Q000737:chemistry*; D051379:Mice; D008915:Minute Virus of Mice / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D018741:Replication Origin; D016615:Telomere; D017361:Viral Nonstructural Proteins / Q000378:metabolism*
D004279:DNA, Viral; C081367:NS1 protein, minute virus of mice; D017361:Viral Nonstructural Proteins; D000255:Adenosine Triphosphate