Faculty Collaboration Database

The A20R protein is a stoichiometric component of the processive form of vaccinia virus DNA polymerase. J Virol 2001 Dec;75(24):12298-307

Date

11/17/2001

Pubmed ID

11711620

Pubmed Central ID

PMC116126

DOI

10.1128/JVI.75.24.12298-12307.2001

Scopus ID

2-s2.0-0035198155 (requires institutional sign-in at Scopus site)   56 Citations

Abstract

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 MgCl(2) 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 approximately 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 approximately 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).

Author List

Klemperer N, McDonald W, Boyle K, Unger B, Traktman P

Author

Paula Traktman Duncan PhD Emeritus Professor in the Microbiology and Immunology department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Amino Acid Sequence
Chromatography
DNA-Directed DNA Polymerase
HeLa Cells
Humans
Immune Sera
Molecular Sequence Data
Molecular Weight
Viral Proteins