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

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Summary

RESULTS

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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Polymerase
DISCUSSION
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