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Journal of Virology, November 1998, p. 8477-8484, Vol. 72, No. 11
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
High-Mobility Group 1/2 Proteins Are Essential for
Initiating Rolling-Circle-Type DNA Replication at a Parvovirus
Hairpin Origin
Susan F.
Cotmore1 and
Peter
Tattersall1,2,*
Departments of Laboratory
Medicine1 and
Genetics,2 Yale University School of
Medicine, New Haven, Connecticut 06510
Received 24 March 1998/Accepted 30 July 1998
Rolling-circle replication is initiated by a replicon-encoded
endonuclease which introduces a single-strand nick into specific origin
sequences, becoming covalently attached to the 5' end of the DNA at the
nick and providing a 3' hydroxyl to prime unidirectional, leading-strand synthesis. Parvoviruses, such as minute virus of mice
(MVM), have adapted this mechanism to amplify their linear single-stranded genomes by using hairpin telomeres which sequentially unfold and refold to shuttle the replication fork back and forth along
the genome, creating a continuous, multimeric DNA strand. The viral
initiator protein, NS1, then excises individual genomes from this
continuum by nicking and reinitiating synthesis at specific origins
present within the hairpin sequences. Using in vitro assays to study
ATP-dependent initiation within the right-hand (5') MVM hairpin, we
have characterized a HeLa cell factor which is absolutely required to
allow NS1 to nick this origin. Unlike parvovirus initiation factor
(PIF), the cellular complex which activates NS1 endonuclease activity
at the left-hand (3') viral origin, the host factor which activates the
right-hand hairpin elutes from phosphocellulose in high salt, has a
molecular mass of around 25 kDa, and appears to bind preferentially to
structured DNA, suggesting that it might be a member of the
high-mobility group 1/2 (HMG1/2) protein family. This prediction was
confirmed by showing that purified calf thymus HMG1 and recombinant
human HMG1 or murine HMG2 could each substitute for the HeLa factor,
activating the NS1 endonuclease in an origin-specific nicking reaction.
*
Corresponding author. Mailing address: Department of
Laboratory Medicine, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06510. Phone: (203) 785-4586. Fax: (203) 688-7340. E-mail: Peter_Tattersall{at}QM.Yale.edu.
Journal of Virology, November 1998, p. 8477-8484, Vol. 72, No. 11
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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