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

doi:10.1128/JVI.75.24.12298-12307.2001

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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-templatebinding 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-infectedcells contain a highly processive form of DNA polymerase, ableto catalyze the replication of a 7-kb template per binding eventunder similar conditions. To study this holoenzyme, we were interestedin purifying and characterizing the vaccinia virus processivityfactor (VPF). Our previous studies indicated that VPF is expressedearly 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 chromatographicpurification procedure, in which a prominent ~45-kDa band wasfound to copurify with processive polymerase activity. This specieswas identified as the product of the A20 gene. By use of recombinantviruses that direct the overexpression of A20 and/or the DNA polymerase,we verified the physical interaction between the two proteinsin coimmunoprecipitation experiments. We also demonstrated thatsimultaneous overexpression of A20 and the DNA polymerase leadsto a specific and robust increase in levels of processive polymeraseactivity. Taken together, we conclude that the A20 gene encodesa component of the processive DNA polymerase complex. Geneticdata that further support this conclusion are presented in theaccompanying report, which documents that temperature-sensitivemutants with lesions in the A20 gene have a DNA phenotype that correlates with a deficit in processive polymeraseactivity (A. Punjabi et al, J. Virol. 75:12308-12318,2001).

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

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