Enzymatic properties of viral replication complexes isolated from adenovirus type 2-infected HeLa cell nuclei

Abstract

When HeLa cell nuclei, isolated 17 h after infection with adenovirus type 2 (Ad2), were extracted with 200 mM ammonium sulfate, Ad2 nucleoprotein complexes were selectively released. These complexes contained a DNA polymerase activity that corresponded to DNA polymerase molecules actively engaged in Ad2 DNA replication. Under our high-salt (200 mM ammonium sulfate) incubation conditions, where no reinitiation occurred, full-length Ad2 DNA chains were synthesized by elongation of chains that had been initiated in vivo. This conclusion was further supported by density labeling experiments indicating that the in vitro DNA synthesis was semiconservative. Evidence is presented suggesting that at least part of the DNA polymerase molecules engaged in Ad2 DNA replication belong to the gamma class.

Adenovirus DNA template for late transcription is not a replicative intermediate

“…Cells were infected with Ad2 at a multiplicity of 100 PFU/cell and labeled with ["C]thymidine (53.9 mCi/mmol, 10 nCi/ml) or [3H]thymidine (25 Ci/mmol, 2 ,uCi/ml) from 6 to 17 h postinfection. Nuclei were purified and extracted with 200 mM ammonium sulfate to obtain fraction S200 (corresponding to the supernatant after extraction) as described elsewhere (4,23).…”

“…Determination of DNA polymerase and RNA polymerase activities. The standard incubation conditions for DNA polymerase and RNA polymerase activities were essentially as described previously (4,23). DNA polymerase activity was assayed at 30°C in an incubation medium containing 200 mM ammonium sulfate; 40 mM Tris-hydrochloride (pH 7.9); 50 ILM each dATP, dGTP, and dTTP; 5 mM MgCl2; and [a-32P]dCTP or [3H]dCTP.…”

“…We have previously shown that, 17 h postinfection, treatment of Ad2-infected HeLa cell nuclei with 200 mM ammonium sulfate selectively releases viral nucleoprotein complexes (23). Among these complexes, both viral transcription and replication complexes are present (4,23). In the present study we have used our system in an attempt to answer the question of whether transcription and replication take place on the same or on different adenovirus DNA molecules.…”

The DNA polymerases associated with the adenovirus type 2 replication complex: effect of 2′-3′-dideoxythymidine-5′-triphosphate on viral DNA synthesis

“…Other in vitro Ad DNA replication systems have been isolated frcm nuclei of infected cells (6,7,8). Each of these systems is able to incorporate deoxynucleotide triphosphates into DNA molecules which had been ¢ Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England initiated in vivo.…”

Erratum

“…A function of DNA polymerase {3 in viral DNA chain growth is less likely since only a and "Y enzymes are found in replication complexes capable of elongation s . 6 and since adenovirus DNA synthesis is sensitive to low concentrations of N -ethylmale imide, which do not inhibit DNA polymerase {3 activity. The function of DNA polymerase "Y in the cell is unknown.…”