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Journal of Virology, December 2001, p. 12298-12307, Vol. 75, No. 24
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.24.12298-12307.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

The A20R Protein Is a Stoichiometric Component of the Processive Form of Vaccinia Virus DNA Polymerase

Nancy Klemperer,1 William McDonald,1 Kathleen Boyle,2 Beth Unger,2 and Paula Traktman1,2,*

Department of Cell Biology and Anatomy, Weill Medical College of Cornell University, New York, New York 10021,1 and Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 532262

Received 16 July 2001/Accepted 10 September 2001

In vitro analysis of the catalytic DNA polymerase encoded by vaccinia virus has demonstrated that it is innately distributive, catalyzing the addition of <10 nucleotides per primer-template binding event in the presence of 8 mM MgCl2 or 40 mM NaCl (W. F. McDonald and P. Traktman, J. Biol. Chem. 269:31190-31197, 1994). In contrast, cytoplasmic extracts isolated from vaccinia virus-infected cells contain a highly processive form of DNA polymerase, able to catalyze the replication of a 7-kb template per binding event under similar conditions. To study this holoenzyme, we were interested in purifying and characterizing the vaccinia virus processivity factor (VPF). Our previous studies indicated that VPF is expressed early after infection and has a native molecular mass of ~48 kDa (W. F. McDonald, N. Klemperer, and P. Traktman, Virology 234:168-175, 1997). Using these criteria, we established a six-step chromatographic purification procedure, in which a prominent ~45-kDa band was found to copurify with processive polymerase activity. This species was identified as the product of the A20 gene. By use of recombinant viruses that direct the overexpression of A20 and/or the DNA polymerase, we verified the physical interaction between the two proteins in coimmunoprecipitation experiments. We also demonstrated that simultaneous overexpression of A20 and the DNA polymerase leads to a specific and robust increase in levels of processive polymerase activity. Taken together, we conclude that the A20 gene encodes a component of the processive DNA polymerase complex. Genetic data that further support this conclusion are presented in the accompanying report, which documents that temperature-sensitive mutants with lesions in the A20 gene have a DNA- phenotype that correlates with a deficit in processive polymerase activity (A. Punjabi et al, J. Virol. 75:12308-12318, 2001).

* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, 8701 Watertown Plank Rd., Rm. BSB-273, Milwaukee, WI 53226. Phone: (414) 456-8253. Fax: (414) 456-6535. E-mail: ptrakt{at}mcw.edu.

Journal of Virology, December 2001, p. 12298-12307, Vol. 75, No. 24
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.24.12298-12307.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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