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Journal of Virology, December 1998, p. 9771-9781, Vol. 72, No. 12
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

The Replication Protein A Binding Site in Simian Virus 40 (SV40) T Antigen and Its Role in the Initial Steps of SV40 DNA Replication

Klaus Weisshart,1 Poonam Taneja,2 and Ellen Fanning2,*

Institute for Molecular Biotechnology, 07745 Jena, Germany,1 and Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, and Vanderbilt Cancer Center, Nashville, Tennessee 37232-68382

Received 6 July 1998/Accepted 4 September 1998

Physical interactions of simian virus 40 (SV40) large tumor (T) antigen with cellular DNA polymerase alpha -primase (Pol/Prim) and replication protein A (RPA) appear to be responsible for multiple functional interactions among these proteins that are required for initiation of viral DNA replication at the origin, as well as during lagging-strand synthesis. In this study, we mapped an RPA binding site in T antigen (residues 164 to 249) that is embedded within the DNA binding domain of T antigen. Two monoclonal antibodies whose epitopes map within this region specifically interfered with RPA binding to T antigen but did not affect T-antigen binding to origin DNA or Pol/Prim, ATPase, or DNA helicase activity and had only a modest effect on origin DNA unwinding, suggesting that they could be used to test the functional importance of this RPA binding site in the initiation of viral DNA replication. To rule out a possible effect of these antibodies on origin DNA unwinding, we used a two-step initiation reaction in which an underwound template was first generated in the absence of primer synthesis. In the second step, primer synthesis was monitored with or without the antibodies. Alternatively, an underwound primed template was formed in the first step, and primer elongation was tested with or without antibodies in the second step. The results show that the antibodies specifically inhibited both primer synthesis and primer elongation, demonstrating that this RPA binding site in T antigen plays an essential role in both events.

* Corresponding author. Mailing address: Department of Molecular Biology, Vanderbilt University, Box 1820 B, Nashville, TN 37235. Phone: (615) 343-5677. Fax: (615) 343-6707. E-mail: FANNINE{at}ctrvax.vanderbilt.edu.

Journal of Virology, December 1998, p. 9771-9781, Vol. 72, No. 12
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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