Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1993 Jan;67(1):265–276. doi: 10.1128/jvi.67.1.265-276.1993

Analysis of the adenovirus type 5 terminal protein precursor and DNA polymerase by linker insertion mutagenesis.

D J Roovers 1, F M van der Lee 1, J van der Wees 1, J S Sussenbach 1
PMCID: PMC237360  PMID: 8416372

Abstract

A series of adenovirus type 5 precursor terminal protein (pTP) and DNA polymerase (Ad pol) genes with linker insertion mutations were separately introduced into the vaccinia virus genome under the control of a late vaccinia virus promoter. The recombinant viruses were used for overexpression of the mutant genes in HeLa cells. In total, 22 different mutant pTP and 10 different Ad pol vaccinia virus recombinants were constructed, including some that expressed carboxyl-terminus-truncated forms of both proteins and one that produced the mutant H5ts149 Ad pol. To investigate the structure-function relationships of both proteins, extracts from cells infected with the recombinant viruses were tested for in vitro complementation of the initiation and elongation steps in adenovirus DNA replication. The results were in accordance with those of earlier in vivo experiments with these insertion mutants and indicate that multiple regions of both proteins are essential for adenovirus DNA replication. The carboxyl termini of both pTP and Ad pol were shown to be essential for proper functioning of these proteins during initiation of adenovirus DNA replication. Three different DNA replication-negative pTP mutants were shown to have residual activity in the initiation assay, suggesting not only that pTP is required for initiation but also that it may play a role in DNA replication after the deoxycytidylation step.

Full text

PDF
265

Images in this article

269Image
on p.269
269Image
on p.269
270Image
on p.270
270Image
on p.270
271Image
on p.271
271Image
on p.271

Selected References

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

  1. Bernad A., Blanco L., Lázaro J. M., Martín G., Salas M. A conserved 3'----5' exonuclease active site in prokaryotic and eukaryotic DNA polymerases. Cell. 1989 Oct 6;59(1):219–228. doi: 10.1016/0092-8674(89)90883-0. [DOI] [PubMed] [Google Scholar]
  2. Bernad A., Lázaro J. M., Salas M., Blanco L. The highly conserved amino acid sequence motif Tyr-Gly-Asp-Thr-Asp-Ser in alpha-like DNA polymerases is required by phage phi 29 DNA polymerase for protein-primed initiation and polymerization. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4610–4614. doi: 10.1073/pnas.87.12.4610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bertholet C., Drillien R., Wittek R. One hundred base pairs of 5' flanking sequence of a vaccinia virus late gene are sufficient to temporally regulate late transcription. Proc Natl Acad Sci U S A. 1985 Apr;82(7):2096–2100. doi: 10.1073/pnas.82.7.2096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Challberg M. D., Desiderio S. V., Kelly T. J., Jr Adenovirus DNA replication in vitro: characterization of a protein covalently linked to nascent DNA strands. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5105–5109. doi: 10.1073/pnas.77.9.5105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Challberg M. D., Kelly T. J., Jr Adenovirus DNA replication in vitro: origin and direction of daughter strand synthesis. J Mol Biol. 1979 Dec 25;135(4):999–1012. doi: 10.1016/0022-2836(79)90524-2. [DOI] [PubMed] [Google Scholar]
  8. Challberg M. D., Kelly T. J., Jr Processing of the adenovirus terminal protein. J Virol. 1981 Apr;38(1):272–277. doi: 10.1128/jvi.38.1.272-277.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chen M., Horwitz M. S. Dissection of functional domains of adenovirus DNA polymerase by linker-insertion mutagenesis. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6116–6120. doi: 10.1073/pnas.86.16.6116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chen M., Mermod N., Horwitz M. S. Protein-protein interactions between adenovirus DNA polymerase and nuclear factor I mediate formation of the DNA replication preinitiation complex. J Biol Chem. 1990 Oct 25;265(30):18634–18642. [PubMed] [Google Scholar]
  11. Drillien R., Spehner D. Physical mapping of vaccinia virus temperature-sensitive mutations. Virology. 1983 Dec;131(2):385–393. doi: 10.1016/0042-6822(83)90506-8. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  14. Field J., Gronostajski R. M., Hurwitz J. Properties of the adenovirus DNA polymerase. J Biol Chem. 1984 Aug 10;259(15):9487–9495. [PubMed] [Google Scholar]
  15. Flores R. A rapid and reproducible assay for quantitative estimation of proteins using bromophenol blue. Anal Biochem. 1978 Aug 1;88(2):605–611. doi: 10.1016/0003-2697(78)90462-1. [DOI] [PubMed] [Google Scholar]
  16. Fredman J. N., Pettit S. C., Horwitz M. S., Engler J. A. Linker insertion mutations in the adenovirus preterminal protein that affect DNA replication activity in vivo and in vitro. J Virol. 1991 Sep;65(9):4591–4597. doi: 10.1128/jvi.65.9.4591-4597.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Freimuth P. I., Ginsberg H. S. Codon insertion mutants of the adenovirus terminal protein. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7816–7820. doi: 10.1073/pnas.83.20.7816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Friefeld B. R., Lichy J. H., Hurwitz J., Horwitz M. S. Evidence for an altered adenovirus DNA polymerase in cells infected with the mutant H5ts149. Proc Natl Acad Sci U S A. 1983 Mar;80(6):1589–1593. doi: 10.1073/pnas.80.6.1589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Guggenheimer R. A., Nagata K., Kenny M., Hurwitz J. Protein-primed replication of plasmids containing the terminus of the adenovirus genome. II. Purification and characterization of a host protein required for the replication of DNA templates devoid of the terminal protein. J Biol Chem. 1984 Jun 25;259(12):7815–7825. [PubMed] [Google Scholar]
  20. Hsieh J. C., Yoo S. K., Ito J. An essential arginine residue for initiation of protein-primed DNA replication. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8665–8669. doi: 10.1073/pnas.87.21.8665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hänggi M., Bannwarth W., Stunnenberg H. G. Conserved TAAAT motif in vaccinia virus late promoters: overlapping TATA box and site of transcription initiation. EMBO J. 1986 May;5(5):1071–1076. doi: 10.1002/j.1460-2075.1986.tb04324.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Joung I., Horwitz M. S., Engler J. A. Mutagenesis of conserved region I in the DNA polymerase from human adenovirus serotype 2. Virology. 1991 Sep;184(1):235–241. doi: 10.1016/0042-6822(91)90840-8. [DOI] [PubMed] [Google Scholar]
  23. Kenny M. K., Balogh L. A., Hurwitz J. Initiation of adenovirus DNA replication. I. Mechanism of action of a host protein required for replication of adenovirus DNA templates devoid of the terminal protein. J Biol Chem. 1988 Jul 15;263(20):9801–9808. [PubMed] [Google Scholar]
  24. Kenny M. K., Hurwitz J. Initiation of adenovirus DNA replication. II. Structural requirements using synthetic oligonucleotide adenovirus templates. J Biol Chem. 1988 Jul 15;263(20):9809–9817. [PubMed] [Google Scholar]
  25. Kwant M. M., van der Vliet P. C. Differential effect of aphidicolin on adenovirus DNA synthesis and cellular DNA synthesis. Nucleic Acids Res. 1980 Sep 11;8(17):3993–4007. doi: 10.1093/nar/8.17.3993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  27. Landschulz W. H., Johnson P. F., McKnight S. L. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science. 1988 Jun 24;240(4860):1759–1764. doi: 10.1126/science.3289117. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Lindenbaum J. O., Field J., Hurwitz J. The adenovirus DNA binding protein and adenovirus DNA polymerase interact to catalyze elongation of primed DNA templates. J Biol Chem. 1986 Aug 5;261(22):10218–10227. [PubMed] [Google Scholar]
  30. 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]
  31. Nagata K., Guggenheimer R. A., Hurwitz J. Adenovirus DNA replication in vitro: synthesis of full-length DNA with purified proteins. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4266–4270. doi: 10.1073/pnas.80.14.4266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pettit S. C., Horwitz M. S., Engler J. A. Adenovirus preterminal protein synthesized in COS cells from cloned DNA is active in DNA replication in vitro. J Virol. 1988 Feb;62(2):496–500. doi: 10.1128/jvi.62.2.496-500.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Pettit S. C., Horwitz M. S., Engler J. A. Mutations of the precursor to the terminal protein of adenovirus serotypes 2 and 5. J Virol. 1989 Dec;63(12):5244–5250. doi: 10.1128/jvi.63.12.5244-5250.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Pruijn G. J., Kusters H. G., Gmelig Meyling F. H., van der Vliet P. C. Inhibition of adenovirus DNA replication in vitro by autoimmune sera. Eur J Biochem. 1986 Jan 15;154(2):363–370. doi: 10.1111/j.1432-1033.1986.tb09406.x. [DOI] [PubMed] [Google Scholar]
  35. Pruijn G. J., van Driel W., van der Vliet P. C. Nuclear factor III, a novel sequence-specific DNA-binding protein from HeLa cells stimulating adenovirus DNA replication. Nature. 1986 Aug 14;322(6080):656–659. doi: 10.1038/322656a0. [DOI] [PubMed] [Google Scholar]
  36. Rijnders A. W., Van Bergen B. G., Van der Vliet P. C., Sussenbach J. S. Immunological characterization of the role of adenovirus terminal protein in viral DNA replication. Virology. 1983 Dec;131(2):287–295. doi: 10.1016/0042-6822(83)90497-x. [DOI] [PubMed] [Google Scholar]
  37. Rijnders A. W., van Bergen B. G., van der Vliet P. C., Sussenbach J. S. Specific binding of the adenovirus terminal protein precursor-DNA polymerase complex to the origin of DNA replication. Nucleic Acids Res. 1983 Dec 20;11(24):8777–8789. doi: 10.1093/nar/11.24.8777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Roovers D. J., Overman P. F., Chen X. Q., Sussenbach J. S. Linker mutation scanning of the genes encoding the adenovirus type 5 terminal protein precursor and DNA polymerase. Virology. 1991 Jan;180(1):273–284. doi: 10.1016/0042-6822(91)90032-7. [DOI] [PubMed] [Google Scholar]
  39. Schaack J., Ho W. Y., Freimuth P., Shenk T. Adenovirus terminal protein mediates both nuclear matrix association and efficient transcription of adenovirus DNA. Genes Dev. 1990 Jul;4(7):1197–1208. doi: 10.1101/gad.4.7.1197. [DOI] [PubMed] [Google Scholar]
  40. Shu L. M., Horwitz M. S., Engler J. A. Expression of enzymatically active adenovirus DNA polymerase from cloned DNA requires sequences upstream of the main open reading frame. Virology. 1987 Dec;161(2):520–526. doi: 10.1016/0042-6822(87)90146-2. [DOI] [PubMed] [Google Scholar]
  41. Shu L., Pettit S. C., Engler J. A. The precise structure and coding capacity of mRNAs from early region 2B of human adenovirus serotype 2. Virology. 1988 Aug;165(2):348–356. doi: 10.1016/0042-6822(88)90579-x. [DOI] [PubMed] [Google Scholar]
  42. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  43. Stillman B. W. Adenovirus DNA replication in vitro: a protein linked to the 5' end of nascent DNA strands. J Virol. 1981 Jan;37(1):139–147. doi: 10.1128/jvi.37.1.139-147.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Stillman B. W., Lewis J. B., Chow L. T., Mathews M. B., Smart J. E. Identification of the gene and mRNA for the adenovirus terminal protein precursor. Cell. 1981 Feb;23(2):497–508. doi: 10.1016/0092-8674(81)90145-8. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. Stunnenberg H. G., Lange H., Philipson L., van Miltenburg R. T., van der Vliet P. C. High expression of functional adenovirus DNA polymerase and precursor terminal protein using recombinant vaccinia virus. Nucleic Acids Res. 1988 Mar 25;16(6):2431–2444. doi: 10.1093/nar/16.6.2431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Sussenbach J. S., van der Vliet P. C. The mechanism of adenovirus DNA replication and the characterization of replication proteins. Curr Top Microbiol Immunol. 1984;109:53–73. doi: 10.1007/978-3-642-69460-8_2. [DOI] [PubMed] [Google Scholar]
  48. Temperley S. M., Hay R. T. Recognition of the adenovirus type 2 origin of DNA replication by the virally encoded DNA polymerase and preterminal proteins. EMBO J. 1992 Feb;11(2):761–768. doi: 10.1002/j.1460-2075.1992.tb05109.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Webster A., Russell S., Talbot P., Russell W. C., Kemp G. D. Characterization of the adenovirus proteinase: substrate specificity. J Gen Virol. 1989 Dec;70(Pt 12):3225–3234. doi: 10.1099/0022-1317-70-12-3225. [DOI] [PubMed] [Google Scholar]
  50. Wong S. W., Wahl A. F., Yuan P. M., Arai N., Pearson B. E., Arai K., Korn D., Hunkapiller M. W., Wang T. S. Human DNA polymerase alpha gene expression is cell proliferation dependent and its primary structure is similar to both prokaryotic and eukaryotic replicative DNA polymerases. EMBO J. 1988 Jan;7(1):37–47. doi: 10.1002/j.1460-2075.1988.tb02781.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Zaballos A., Salas M. Functional domains in the bacteriophage phi 29 terminal protein for interaction with the phi 29 DNA polymerase and with DNA. Nucleic Acids Res. 1989 Dec 25;17(24):10353–10366. doi: 10.1093/nar/17.24.10353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Zhao L. J., Padmanabhan R. Nuclear transport of adenovirus DNA polymerase is facilitated by interaction with preterminal protein. Cell. 1988 Dec 23;55(6):1005–1015. doi: 10.1016/0092-8674(88)90245-0. [DOI] [PubMed] [Google Scholar]
  53. Zhao L. J., Padmanabhan R. Three basic regions in adenovirus DNA polymerase interact differentially depending on the protein context to function as bipartite nuclear localization signals. New Biol. 1991 Nov;3(11):1074–1088. [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES