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Journal of Virology, January 2000, p. 744-754, Vol. 74, No. 2
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

A Role for Single-Stranded Templates in Cell-Free Adeno-Associated Virus DNA Replication

Peter Ward* and R. Michael Linden

Institute for Gene Therapy and Molecular Medicine, Mt. Sinai School of Medicine, New York, New York 10029

Received 17 August 1999/Accepted 15 October 1999

Assays have been described in which duplex adeno-associated virus (AAV) DNA can be replicated in HeLa cell extracts with exogenous AAV Rep protein. These assays appear to mimic the AAV DNA replication that occurs in the cell, including the ability of extracts from adenovirus (Ad)-infected cells to replicate duplex AAV DNA templates more efficiently than extracts from uninfected cells can. We showed previously that the Ad-infected extract was able to support a more processive replication than the uninfected extract. When the Ad single-stranded DNA binding protein (Ad-DBP) was added to an uninfected extract, DNA replication became processive. Based on a strand displacement replication model, we hypothesized that the Ad-DBP was stabilizing the displaced single-stranded DNA during strand displacement replication. In this report, we show that in Ad-infected extracts most of the newly replicated duplex DNA is converted into a single-stranded form shortly after synthesis. Using the results of assays for the replication of single-stranded AAV DNA, we show that these single-stranded molecules serve as templates for additional replication. In addition, we identify a class of molecules which are likely to be intermediates of replication on single-stranded templates. We discuss a possible role for replication of single-stranded molecules in the infected cell.

* Corresponding author. Mailing address: Institute for Gene Therapy and Molecular Medicine, Mt. Sinai School of Medicine, Box 1496, One Gustave L. Levy Place, New York, NY 10029-6574. Phone: (212) 659-8247. Fax: (212) 849-2437. E-mail: wardp01{at}doc.mssm.edu.

Journal of Virology, January 2000, p. 744-754, Vol. 74, No. 2
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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