INVOLVEMENT OF EUKARYOTIC DEOXYRIBONUCLEIC-ACID POLYMERASE-ALPHA AND POLYMERASE-GAMMA IN THE REPLICATION OF CELLULAR AND VIRAL DEOXYRIBONUCLEIC-ACID

In an effort to identify the deoxyribonucleic acid (DNA) polymerase activities responsible for mammalian viral and cellular DNA replication, the effect of DNA synthesis inhibitors on isolated DNA polymerases was compared with their effects on viral and cellular DNA replication in vitro. DNA polymerase a, simian virus 40 (SV40) DNA replication in nuclear extracts, and CV-1 cell (the host for SV40) DNA replication in isolated nuclei all responded to DNA synthesis inhibitors in a quantitatively similar manner: they were relatively insensitive to 2',3'-dideoxythymidine 5'-triphosphate (d2TTP), but completely inhibited by aphidicolin, 1 -β-d-arabinofuranosylcytosine 5'-triphosphate (araCTP), and N-ethylmaleimide. In comparison, DNA polymerases β and γ were inhibited by d2TTP but insensitive to aphidicolin and 20-30 times less sensitive to araCTP than DNA polymerase a. Herpes simplex virus type 1 (HSV-1) DNA polymerase and DNA polymerase α were the only enzymes tested that were relatively insensitive to d2TTP; DNA polymerases β and γ, phages T4 and T7 DNA polymerases, and Escherichia coli DNA polymerase I were 100-250 times more sensitive. The results with d2TTP were independent of enzyme concentration, primer-template concentration, primer-template choice, and the labeled dNTP. A specific requirement for DNA ponlymerase α in the replication of SV40 DNA was demonstrated by the fact that DNA polymerase a was required, in addition to other cytosol proteins, to reconstitute SV40 DNA replication activity in N-ethylmaleimide-inactivated nuclear extracts containing replicating SV40 chromosomes. DNA polymerases β and γ did not substitute for DNA polymerase α. In contrast to SV40 and CV-1 DNA replication, adenovirus type 2 (Ad-2) DNA replication in isolated nuclei was inhibited by d2TTP to the same extent as γ-polymerase. Ad-2 DNA replication was also inhibited by aphidicolin to the same extent asα-polymerase. Synthesis of CV-1 DNA, SV40 DNA, and HSV-1 DNA in intact CV-1 cells was inhibited by aphidicolin. Ad-2 DNA replication was also inhibited, but only at a 100-fold higher concentration. We found no effect of 2',3'-dideoxythymidine (d2Thd) on cellular or viral DNA replication in spite of the fact that Ad-2 DNA replication in isolated nuclei was inhibited 50% by a ratio of d2TTP/dTTP of 0.02. This was due to the inability of CV-1 and Hela cells to phosphorylate d2Thd to d2TTP. These data are consistent with the hypothesis that DNA polymerase α is the only DNA polymerase involved in replicating SV40 DNA and CV-1 DNA and that Ad-2 DNA replication involves both DNA polymerases γ and α. © 1979, American Chemical Society. All rights reserved.