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.
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).
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