Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1983 Mar;80(6):1589–1593. doi: 10.1073/pnas.80.6.1589

Evidence for an altered adenovirus DNA polymerase in cells infected with the mutant H5ts149

Beth R Friefeld *, Jack H Lichy †, Jerard Hurwitz †, Marshall S Horwitz *,‡
PMCID: PMC393647  PMID: 6572922

Abstract

The N complementation group of adenovirus (Ad) serotype 5 mutants, which are temperature sensitive for viral DNA synthesis in vivo, has been used to study a 140,000-dalton DNA polymerase (Pol) that copurified with the 80,000-dalton terminal protein precursor (pTP). Extracts prepared from HeLa cells infected with the N group mutant H5ts149 at nonpermissive temperature were unable to synthesize viral DNA. The defect in these extracts was specifically reversed by addition of the Pol purified from wild-type Ad-infected cytosol. Addition of the pTP, free of the Pol, did not restore replicative activity to H5ts149 extracts. The reactions studied depend on the presence of the DNA template and include the initiation reaction (the covalent attachment of dCMP to the pTP) and the selective replication of Ad DNA restriction endonuclease fragments containing the origin sequences. Glycerol gradient sedimentation showed that a replicative activity representing the pTP-Pol complex was greatly reduced in H5ts149 extracts as compared with wild-type extracts, suggesting some alteration in the mutant. A pool of pTP free of Pol was detected on these gradients in extracts from both wild-type and H5ts149-infected cells. In addition, the initiation and elongation of Ad DNA catalyzed by H5ts149 extracts prepared from cells grown at permissive temperatures was more labile to urea inactivation than extracts prepared from cells infected with wild-type virus. These results, considered together with the mapping of the H5ts149 mutation within an open reading frame approximately large enough to code for the 140,000-dalton DNA polymerase [Gingeras, T. R., Sciaky, D., Gelinas, R. E., Bing-Dong, J., Yen, C. E., Kelly, M. M., Bullock, P. A., Parsons, B. L., O'Neill, K. E. & Roberts, R. J. (1982) J. Biol. Chem. 257, 13475-13491; Alestrom, P., Akusjarui, G., Pettersson, M. & Pettersson, U. (1982) J. Biol. Chem. 257, 13492-13498], suggest that the Pol is a virally encoded protein, as is the pTP.

Keywords: DNA synthesis, temperature-sensitive mutation

Full text

PDF
1589

Images in this article

1591Image
on p.1591
1591Image
on p.1591
1591Image
on p.1591
1592Image
on p.1592

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aleström P., Akusjärvi G., Pettersson M., Pettersson U. DNA sequence analysis of the region encoding the terminal protein and the hypothetical N-gene product of adenovirus type 2. J Biol Chem. 1982 Nov 25;257(22):13492–13498. [PubMed] [Google Scholar]
  2. Challberg M. D., Kelly T. J., Jr Adenovirus DNA replication in vitro. Proc Natl Acad Sci U S A. 1979 Feb;76(2):655–659. doi: 10.1073/pnas.76.2.655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Challberg M. D., Ostrove J. M., Kelly T. J., Jr Initiation of adenovirus DNA replication: detection of covalent complexes between nucleotide and the 80-kilodalton terminal protein. J Virol. 1982 Jan;41(1):265–270. doi: 10.1128/jvi.41.1.265-270.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Enomoto T., Lichy J. H., Ikeda J. E., Hurwitz J. Adenovirus DNA replication in vitro: purification of the terminal protein in a functional form. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6779–6783. doi: 10.1073/pnas.78.11.6779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ensinger M. J., Ginsberg H. S. Selection and preliminary characterization of temperature-sensitive mutants of type 5 adenovirus. J Virol. 1972 Sep;10(3):328–339. doi: 10.1128/jvi.10.3.328-339.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Galos R. S., Williams J., Binger M. H., Flint S. J. Location of additional early gene sequences in the adenoviral chromosome. Cell. 1979 Aug;17(4):945–956. doi: 10.1016/0092-8674(79)90334-9. [DOI] [PubMed] [Google Scholar]
  7. Gingeras T. R., Sciaky D., Gelinas R. E., Bing-Dong J., Yen C. E., Kelly M. M., Bullock P. A., Parsons B. L., O'Neill K. E., Roberts R. J. Nucleotide sequences from the adenovirus-2 genome. J Biol Chem. 1982 Nov 25;257(22):13475–13491. [PubMed] [Google Scholar]
  8. Horwitz M. S., Ariga H. Multiple rounds of adenovirus DNA synthesis in vitro. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1476–1480. doi: 10.1073/pnas.78.3.1476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Horwitz M. S. Temperature-sensitive replication of H5ts125 adenovirus DNA in vitro. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4291–4295. doi: 10.1073/pnas.75.9.4291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ikeda J. E., Enomoto T., Hurwitz J. Replication of adenovirus DNA-protein complex with purified proteins. Proc Natl Acad Sci U S A. 1981 Feb;78(2):884–888. doi: 10.1073/pnas.78.2.884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ikeda J. E., Longiaru M., Horwitz M. S., Hurwitz J. Elongation of primed DNA templates by eukaryotic DNA polymerases. Proc Natl Acad Sci U S A. 1980 Oct;77(10):5827–5831. doi: 10.1073/pnas.77.10.5827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kaplan L. M., Ariga H., Hurwitz J., Horwitz M. S. Complementation of the temperature-sensitive defect in H5ts125 adenovirus DNA replication in vitro. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5534–5538. doi: 10.1073/pnas.76.11.5534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lichy J. H., Field J., Horwitz M. S., Hurwitz J. Separation of the adenovirus terminal protein precursor from its associated DNA polymerase: role of both proteins in the initiation of adenovirus DNA replication. Proc Natl Acad Sci U S A. 1982 Sep;79(17):5225–5229. doi: 10.1073/pnas.79.17.5225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lichy J. H., Horwitz M. S., Hurwitz J. Formation of a covalent complex between the 80,000-dalton adenovirus terminal protein and 5'-dCMP in vitro. Proc Natl Acad Sci U S A. 1981 May;78(5):2678–2682. doi: 10.1073/pnas.78.5.2678. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Nagata K., Guggenheimer R. A., Enomoto T., Lichy J. H., Hurwitz J. Adenovirus DNA replication in vitro: identification of a host factor that stimulates synthesis of the preterminal protein-dCMP complex. Proc Natl Acad Sci U S A. 1982 Nov;79(21):6438–6442. doi: 10.1073/pnas.79.21.6438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Sharp P. A., Moore C., Haverty J. L. The infectivity of adenovirus 5 DNA-protein complex. Virology. 1976 Dec;75(2):442–456. doi: 10.1016/0042-6822(76)90042-8. [DOI] [PubMed] [Google Scholar]
  17. Smart J. E., Stillman B. W. Adenovirus terminal protein precursor. Partial amino acid sequence and the site of covalent linkage to virus DNA. J Biol Chem. 1982 Nov 25;257(22):13499–13506. [PubMed] [Google Scholar]
  18. Stillman B. W., Tamanoi F., Mathews M. B. Purification of an adenovirus-coded DNA polymerase that is required for initiation of DNA replication. Cell. 1982 Dec;31(3 Pt 2):613–623. doi: 10.1016/0092-8674(82)90317-8. [DOI] [PubMed] [Google Scholar]
  19. Wilkie N. M., Ustacelebi S., Williams J. F. Characterization of temperature-sensitive mutants of adenovirus type 5: nucleic acid synthesis. Virology. 1973 Feb;51(2):499–503. doi: 10.1016/0042-6822(73)90450-9. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES