Abstract
We have partially purified and characterized two separate DNA polymerase activities associated with Epstein-Barr virus (EB virus). One activity is present in EB virus producer cell lines but not in nonproducer or negative cell lines. It adheres more strongly to DEAE-cellulose than any host cell enzymes, eluting at 210 to 270 mM potassium phosphate buffer. Further elution from phosphocellulose and sedimentation in glycerol gradients yields an enzyme purified 900-fold with an S value of 8.3. The second DNA polymerase activity co-purifies with EB viral particles, elutes at low salt from DEAE-cellulose (40 to 60 mM potassium phosphate buffer) and phosphocellulose (100 mM), and has an S value of 9.5 on glycerol gradient sedimentation. These two enzymes are referred to for convenience as the EB virus-induced DNA polymerase and the EB virion-associated DNA polymerase. The EB virus-induced polymerase can be distinguished from host alpha, beta, and the virion-associated polymerase in 1) being resistant to salt inhibition, 2) having a more basic pH optima in Tris buffer (pH 9.5), and 3) having a 10-fold lower saturating concentration for the activated DNA template. The EB virion-associated polymerase is distinguished from host alpha, beta, and the EB virus-induced polymerase, because it cannot utilize synthetic deoxy- and ribohomopolymer primer-templates in place of the activated calf thymus DNA template in DNA polymerase assays. Neither of the EB virus-associated polymerases can copy the ribohomopolymers dT10poly(rA) or dG12-18(poly(rC) efficiently and therefore can be distinguished from host gamma polymerase and reverse transcriptase. The activity of the EB virus-induced and virion-associated polymerases are unaffected both by antibody to alpha polymerase, and by antiserum with high antibody titers to EB early antigen and viral capsid antigen.
Highlights
Animal cells normally contain three major classes of DNA polymerases: Class 1, a polymerase, a large (7 S), N-ethylmaleimide-sensitive enzyme whose activity varies with cellular growth rates; Class 2, /3 polymerase, a small (3 to 4 S) iV-ethylmaleimide-resistant enzyme whose activity is unaffected by cellular growth rate; and class 3, y polymerase, of intermediate size (6 S), which comprises only 1 to 2% of the total cellular DNA polymerase activity, and can be distinguished from the two major eukaryotic DNA polymerases in its ability to copy ribohomopolymers such as dT1,(A), at a much greater rate than gapped duplex DNA
We have demonstrated the existence of two EB virus-associated DNA polymerase activities
When P3HR-1 is kept in continuous culture, causing a decrease in viral capsid antigen and viral production, the levels of this enzyme decrease in direct correlation with this drop in virus production
Summary
Cellulose columns under high salt conditions in order to remove DNA without loss of enzyme. As can be seen in Fig. lA, P3HR-1 contains a species of DNA polymerase activity which adheres more tenaciously to DEAE-cellulose than any of the host cell polymerases, eluting at greater than 200 InM potassium phosphate. The EB virus-induced DNA polymerase activity that was eluted at 210 to 270 mM potassium phosphate from a DEAEcellulose column such as that in Fig. 1A was pooled, dialyzed against low salt Buffer C, and further chromatographed on a phosphocellulose column. Under these conditions, the EB virus polymerase adheres to phosphocellulose while most of the other proteins in the preparation do not.
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