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Journal of Virology, September 2001, p. 8569-8578, Vol. 75, No. 18
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.18.8569-8578.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Amino Acids 257 to 288 of Mouse p48 Control the Cooperation of Polyomavirus Large T Antigen, Replication Protein A, and DNA Polymerase -Primase To Synthesize DNA In Vitro

Armin R. Kautz, Klaus Weisshart, Annerose Schneider, Frank Grosse, and Heinz-Peter Nasheuer^*

Abteilung Biochemie, Institut für Molekulare Biotechnologie e.V., D-07745 Jena, Germany

Received 16 January 2001/Accepted 14 June 2001

Although p48 is the most conserved subunit of mammalian DNA polymerase -primase (pol-prim), the polypeptide is the major species-specific factor for mouse polyomavirus (PyV) DNA replication. Human and murine p48 contain two regions (A and B) that show significantly lower homology than the rest of the protein. Chimerical human-murine p48 was prepared and coexpressed with three wild-type subunits of pol-prim, and four subunit protein complexes were purified. All enzyme complexes synthesized DNA on single-stranded (ss) DNA and replicated simian virus 40 DNA. Although the recombinant protein complexes physically interacted with PyV T antigen (Tag), we determined that the murine region A mediates the species specificity of PyV DNA replication in vitro. More precisely, the nonconserved phenylalanine 262 of mouse p48 is crucial for this activity, and pol-prim with mutant p48, h-S262F, supports PyV DNA replication in vitro. DNA synthesis on RPA-bound ssDNA revealed that amino acid (aa) 262, aa 266, and aa 273 to 288 are involved in the functional cooperation of RPA, pol-prim, and PyV Tag.

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^* Corresponding author. Mailing address: Institut für Molekulare Biotechnologie e.V., Abt. Biochemie, Beutenbergstr. 11, D-07745 Jena, Germany. Phone: 49-3641-656290. Fax: 49-3641-656288. E-mail: nasheuer{at}imb-jena.de.

Present address: Biomedical Apherese Systeme GmbH, 07745 Jena, Germany.

Present address: Carl Zeiss Jena GmbH, D-07745 Jena, Germany.

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Journal of Virology, September 2001, p. 8569-8578, Vol. 75, No. 18
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.18.8569-8578.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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