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Journal of Virology, June 2002, p. 5835-5845, Vol. 76, No. 11
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.11.5835-5845.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Induction of the Bovine Papillomavirus Origin "Onion Skin"-Type DNA Replication at High E1 Protein Concentrations In Vivo

Andres Männik,¹ Kertu Rünkorg,¹ Nele Jaanson,¹ Mart Ustav,^1,2 and Ene Ustav^2*

Department of Microbiology and Virology, Institute of Molecular and Cell Biology, Estonian Biocentre,¹ Department of Gene Technology, Center of Technology, Tartu University, Tartu, Estonia²

Received 5 July 2001/ Accepted 25 February 2002

We have studied the replication of plasmids composed of bovine papillomavirus type 1 (BPV1) origin of replication and expression cartridges for viral proteins E1 and E2 in hamster and mouse cells. We found that the replication mode changed dramatically at different expression levels of the E1 protein. At high levels of the E1 protein, overreplication of the origin region of the plasmid was observed. Analysis of the replication products by one-dimensional and two-dimensional gel electrophoresis suggested that initially "onion skin"-type replication intermediates were generated, presumably resulting from initiation of the new replication forks before the leading fork completed the synthesis of the DNA on the episomal plasmid. These replication intermediates served as templates for generation of a heterogeneous set of origin region-containing linear fragments by displacement synthesis at the partially replicated plasmid. Additionally, the linear fragments may have been generated by DNA break-up of the onion skin-type intermediates. Analysis of replication products indicated that generated linear fragments recombined and formed concatemers or circular molecules, which presumably were able to replicate in an E1- and E2-dependent fashion. At moderate and low levels of E1, generated by transcription of the E1 open reading frame using weaker promoters, DNA replication was initiated at much lower levels, which allowed elongation of the replication fork starting from the origin to be more balanced and resulted in the generation of full-sized replication products.

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* Corresponding author. Mailing address: Department of Gene Technology, Center of Technology, Tartu University, 23 Riia St., Tartu 51010, Estonia. Phone: 372-7-375041. Fax: 372-7-420286. E-mail: eustav{at}ebc.ee.

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Journal of Virology, June 2002, p. 5835-5845, Vol. 76, No. 11
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.11.5835-5845.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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