This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Klemperer, N.
Right arrow Articles by Traktman, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Klemperer, N.
Right arrow Articles by Traktman, P.

 Previous Article  |  Next Article 

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.


This article has been cited by other articles:

  • Nichols, R. J., Stanitsa, E., Unger, B., Traktman, P. (2008). The Vaccinia Virus Gene I2L Encodes a Membrane Protein with an Essential Role in Virion Entry. J. Virol. 82: 10247-10261 [Abstract] [Full Text]  
  • Hamilton, M. D., Nuara, A. A., Gammon, D. B., Buller, R. M., Evans, D. H. (2007). Duplex strand joining reactions catalyzed by vaccinia virus DNA polymerase. Nucleic Acids Res 35: 143-151 [Abstract] [Full Text]  
  • Kornbluth, R. S., Smee, D. F., Sidwell, R. W., Snarsky, V., Evans, D. H., Hostetler, K. Y. (2006). Mutations in the E9L Polymerase Gene of Cidofovir-Resistant Vaccinia Virus Strain WR Are Associated with the Drug Resistance Phenotype. Antimicrob. Agents Chemother. 50: 4038-4043 [Abstract] [Full Text]  
  • Stanitsa, E. S., Arps, L., Traktman, P. (2006). Vaccinia Virus Uracil DNA Glycosylase Interacts with the A20 Protein to Form a Heterodimeric Processivity Factor for the Viral DNA Polymerase. J. Biol. Chem. 281: 3439-3451 [Abstract] [Full Text]  
  • Magee, W. C., Hostetler, K. Y., Evans, D. H. (2005). Mechanism of Inhibition of Vaccinia Virus DNA Polymerase by Cidofovir Diphosphate. Antimicrob. Agents Chemother. 49: 3153-3162 [Abstract] [Full Text]  
  • Hamilton, M. D., Evans, D. H. (2005). Enzymatic processing of replication and recombination intermediates by the vaccinia virus DNA polymerase. Nucleic Acids Res 33: 2259-2268 [Abstract] [Full Text]  
  • Senkevich, T. G., Ward, B. M., Moss, B. (2004). Vaccinia Virus Entry into Cells Is Dependent on a Virion Surface Protein Encoded by the A28L Gene. J. Virol. 78: 2357-2366 [Abstract] [Full Text]  
  • Boyle, K. A., Traktman, P. (2004). Members of a Novel Family of Mammalian Protein Kinases Complement the DNA-Negative Phenotype of a Vaccinia Virus ts Mutant Defective in the B1 Kinase. J. Virol. 78: 1992-2005 [Abstract] [Full Text]  
  • Scaramozzino, N., Sanz, G., Crance, J. M., Saparbaev, M., Drillien, R., Laval, J., Kavli, B., Garin, D. (2003). Characterisation of the substrate specificity of homogeneous vaccinia virus uracil-DNA glycosylase. Nucleic Acids Res 31: 4950-4957 [Abstract] [Full Text]  
  • De Silva, F. S., Moss, B. (2002). Vaccinia Virus Uracil DNA Glycosylase Has an Essential Role in DNA Synthesis That Is Independent of Its Glycosylase Activity: Catalytic Site Mutations Reduce Virulence but Not Virus Replication in Cultured Cells. J. Virol. 77: 159-166 [Abstract] [Full Text]  
  • Punjabi, A., Boyle, K., DeMasi, J., Grubisha, O., Unger, B., Khanna, M., Traktman, P. (2001). Clustered Charge-to-Alanine Mutagenesis of the Vaccinia Virus A20 Gene: Temperature-Sensitive Mutants Have a DNA-Minus Phenotype and Are Defective in the Production of Processive DNA Polymerase Activity. J. Virol. 75: 12308-12318 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»