Adenovirus DNA replication in vitro: purification of the terminal protein in a functional form.
The 80,000-dalton form of the adenovirus (Ad) terminal protein (pTP) has been purified from Ad-infected HeLa cells. pTP was assayed by its ability to form a covalent complex with dCMP. The protein copurified with an activity that is essential for in vitro Ad DNA replication (Ad protein activity) as well as with a DNA polymerase activity that was distinguished from those of HeLa cell DNA polymerases alpha, beta, and gamma. The Ad protein-associated DNA polymerase activity was detected with activated DNA but not with poly(rA).oligo(dT) as template and was insensitive to aphidicolin and sensitive to N-ethylmaleimide. The Ad protein, DNA polymerase, and pTP-dCMP complex-forming activities sedimented in a glycerol gradient as a single peak with an apparent molecular size of 180,000 daltons. NaDodSO4/polyacrylamide gel analysis of the glycerol gradient fraction showed major bands of 80,000 and 140,000 daltons. The 80,000-dalton band was identified as pTP by comparison of its tryptic peptide map with that of the 55,000-dalton form of the terminal protein, which was purified from Ad virions.
D000260:Adenoviruses, Human / Q000201:enzymology*; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D008970:Molecular Weight; D014764:Viral Proteins / Q000302:isolation & purification / Q000378:metabolism*
D014764:Viral Proteins; D004259:DNA-Directed DNA Polymerase