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

Vaccinia virus exhibits a high degree of genetic and physical autonomy from the host cell, possessing a genome that encodes more than 200 genes and directing a replicative cycle in which DNA replication, gene expression, and morphogenesis take place solely within the cytoplasm of the infected cell. It is therefore likely that the trans-acting functions required for DNA replication are virally encoded, and several laboratories have engaged in studies aimed at identifying and characterizing these functions. The catalytic DNA polymerase of vaccinia virus is encoded by the E9 gene (6, 20, 31). The 116-kDa enzyme possesses both polymerase and proofreading exonuclease activity and retains many of the conserved domains that are found within the replicative polymerases of mammalian and yeast cells, as well as those encoded by other large DNA viruses. In addition, the vaccinia virus enzyme, like the catalytic subunit of these other replicative polymerases, is inherently distributive. Our studies on the purified E9 protein indicate that in the presence of 40 mM NaCl or 8 mM MgCl2, the enzyme catalyzes the addition of 7,000 nt per primer-template binding event. This dichotomy between the distributive behavior of the catalytic subunit of a polymerase and the processive behavior of the replicative holoenzyme is virtually universal in viral, prokaryotic, and eukaryotic systems. Comparative analyses of these systems has revealed several molecular strategies for generating a processive enzyme. The cellular enzymes interact with a toroidal processivity factor that associates topologically, but not directly, with the template DNA. Loading these sliding clamps (a homotrimer of PCNA in eukaryotic cells; a homodimer of the β subunit in Escherichia coli) onto the DNA requires an ATP-hydrolyzing multisubunit clamp loader (replication factor C in eukaryotic cells; the γ complex in E. coli) (27). Bacteriophage T4 also uses a comparable strategy for achieving processivity. Bacteriophage T7 utilizes a novel strategy in which processivity is achieved by interaction of the polymerase with thioredoxin; however, since thioredoxin does not bind to DNA, the mechanism by which processivity is gained remains unclear. A distinct mechanism appears to be used by some eukaryotic viruses. In herpes simplex virus, the UL42 protein binds both to DNA and to the catalytic polymerase, serving to tether the polymerase to the primer-template without inhibiting its ability to translocate along the DNA (1, 15). The UL42 protein is able to confer processivity on the viral DNA polymerase by increasing its rate of association with, and decreasing its rate of disassociation from, the primer-template junction (32). We have previously shown that the factor(s) responsible for conferring processivity on purified vaccinia virus DNA polymerase is present within cytoplasmic extracts prepared from infected, but not uninfected, cells. Moreover, we have demonstrated that accumulation of the factor required only the early phase of gene expression and that the vaccinia virus processivity factor (VPF) appeared to have a native molecular mass of approximately 48 kDa (19). In this report, we describe the purification of a 49-kDa component of the processive polymerase and its identification as the product of the A20 gene. In an accompanying report (28), we present the utilization of clustered charge-to-alanine mutagenesis and transient dominant selection to generate vaccinia viruses containing altered alleles of the A20 gene, and we show that two such viruses have a temperature-sensitive DNA− phenotype that correlates with a defect in processive polymerase activity.