Involvement of both DNA polymerases α and γ in the replication of adenovirus deoxyribonucleic acid in vitro

Abstract

Adenovirus (Ad) DNA replication complexes (RCs) were purified from nuclear extracts of Ad-infected cells by exclusion chromatography, followed by equilibrium centrifugation in discontinuous sucrose-heavy water gradients. The material, which was characterized as the site for synthesis of Ad DNA by its time course of labeling in vivo, was able to elongate, in vitro, viral DNA molecules, the synthesis of which had been initiated in vivo. Endogenous DNA polymerase activity in the Ad RCs was totally inhibited by dideoxythymidine triphosphate (ddTTP) at a ddTTP:dTTP ratio of about 5:100; this suggests that the functional enzyme involved was DNA polymerase γ. The activity of the RCs was also strongly inhibited by 50 μg/ml phosphonoacetic acid (PPAA), and by Ara CTP:dCTP ratios of about 5:1; this suggests that the functional enzyme involved in the elongation activity was DNA polymerase α. Sensitivity of Ad RC's endogenous activity toward aphidicolin was somewhat lower than that reported for purified DNA polymerase α, but definitely much higher than that reported for DNA polymerase γ. Direct determination of the DNA polymerases present in partially purified Ad RCs was done by sucrose gradient centrifugation followed by identification of the enzymes through the use of specific assays. The major DNA polymerase present in Ad RCs was DNA polymerase α. In addition, small amounts of DNA polymerase γ were detected. No DNA polymerase β appeared to be present. These data are consistent with the hypothesis that Ad5 DNA replication involves both DNA polymerases α and γ.