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Journal of Virology, May 2002, p. 5121-5130, Vol. 76, No. 10
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.10.5121-5130.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Mutational Analysis of Simian Virus 40 T-Antigen Primosome Activities in Viral DNA Replication

Robert D. Ott, Yingda Wang, and Ellen Fanning*

Department of Biological Sciences and Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, Tennessee 37232

Received 10 August 2001/ Accepted 4 January 2002

The recruitment of DNA polymerase {alpha}-primase (pol-prim) is a crucial step in the establishment of a functional replication complex in eukaryotic cells, but the mechanism of pol-prim loading and the composition of the eukaryotic primosome are poorly understood. In the model system for simian virus 40 (SV40) DNA replication in vitro, synthesis of RNA primers at the origin of replication requires only the viral tumor (T) antigen, replication protein A (RPA), pol-prim, and topoisomerase I. On RPA-coated single-stranded DNA (ssDNA), T antigen alone mediates priming by pol-prim, constituting a relatively simple primosome. T-antigen activities proposed to participate in its primosome function include DNA helicase and protein-protein interactions with RPA and pol-prim. To test the role of these activities of T antigen in mediating priming by pol-prim, three replication-defective T antigens with mutations in the ATPase or helicase domain have been characterized. All three mutant proteins interacted physically and functionally with RPA and pol-prim and bound ssDNA, and two of them displayed some helicase activity. However, only one of these, 5030, mediated primer synthesis and elongation by pol-prim on RPA-coated ssDNA. The results suggest that a novel activity, present in 5030 T antigen and absent in the other two mutants, is required for T-antigen primosome function.

* Corresponding author. Mailing address: Department of Biological Sciences, Vanderbilt University, 1161 21st Ave. South, Nashville, TN 37232. Phone: (615) 343-5677. Fax: (615) 343-6707. E-mail: fannine{at}ctrvax.vanderbilt.edu.

Journal of Virology, May 2002, p. 5121-5130, Vol. 76, No. 10
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.10.5121-5130.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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