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Journal of Virology, May 2007, p. 4808-4818, Vol. 81, No. 9
0022-538X/07/$08.00+0     doi:10.1128/JVI.02451-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Model for T-Antigen-Dependent Melting of the Simian Virus 40 Core Origin Based on Studies of the Interaction of the Beta-Hairpin with DNA

Anuradha Kumar,¹ Gretchen Meinke,¹ Danielle K. Reese,¹ Stephanie Moine,¹ Paul J. Phelan,¹ Amélie Fradet-Turcotte,² Jacques Archambault,² Andrew Bohm,¹ and Peter A. Bullock^1*

Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111,¹ Institut de Recherches Cliniques de Montreal, 110 Avenue des Pins Ouest, Montreal, Quebec, Canada H2W 1R7²

Received 7 November 2006/ Accepted 30 January 2007

The interaction of simian virus 40 (SV40) T antigen (T-ag) with the viral origin has served as a model for studies of site-specific recognition of a eukaryotic replication origin and the mechanism of DNA unwinding. These studies have revealed that a motif termed the "beta-hairpin" is necessary for assembly of T-ag on the SV40 origin. Herein it is demonstrated that residues at the tip of the "beta-hairpin" are needed to melt the origin-flanking regions and that the T-ag helicase domain selectively assembles around one of the newly generated single strands in a manner that accounts for its 3'-to-5' helicase activity. Furthermore, T-ags mutated at the tip of the "beta-hairpin" are defective for oligomerization on duplex DNA; however, they can assemble on hybrid duplex DNA or single-stranded DNA (ssDNA) substrates provided the strand containing the 3' extension is present. Collectively, these experiments indicate that residues at the tip of the beta-hairpin generate ssDNA in the core origin and that the ssDNA is essential for subsequent oligomerization events.

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* Corresponding author. Mailing address: Department of Biochemistry A703, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Phone: (617) 636-0447. Fax: (617) 636-2409. E-mail: Peter.Bullock{at}tufts.edu

Published ahead of print on 7 February 2007.

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Journal of Virology, May 2007, p. 4808-4818, Vol. 81, No. 9
0022-538X/07/$08.00+0     doi:10.1128/JVI.02451-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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