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. 1986 Jun;6(6):2115–2124. doi: 10.1128/mcb.6.6.2115

Template requirements for in vivo replication of adenovirus DNA.

J A Bernstein, J M Porter, M D Challberg
PMCID: PMC367752  PMID: 3785188

Abstract

The adenovirus (Ad) DNA origin of replication was defined through an analysis of the DNA sequences necessary for the replication of plasmid DNAs with purified viral and cellular proteins. Results from several laboratories have shown that the origin consists of two functionally distinct domains: a 10-base-pair sequence present in the inverted terminal repetition (ITR) of all human serotypes and an adjacent sequence constituting the binding site for a cellular protein, nuclear factor I. To determine whether the same nucleotide sequences are necessary for origin function in vivo, we developed an assay for the replication of plasmid DNAs transfected into Ad5-infected cells. The assay is similar to that described by Hay et al. (J. Mol. Biol. 175:493-510, 1984). With this assay, plasmid DNA replication is dependent upon prior infection of cells with virus and only occurs with linear DNA molecules containing viral terminal sequences at each end. Replicated DNA is resistant to digestion with lambda-exonuclease, suggesting that a protein is covalently bound at both termini. A plasmid containing only the first 67 base pairs of the Ad2 ITR replicates as well as plasmids containing the entire ITR. Deletions or point mutations which reduce the binding of nuclear factor I to DNA in vitro reduce the efficiency of plasmid replication in vivo. A point mutation within the 10-base-pair conserved sequence has a similar effect upon replication. These results suggest that the two sequence domains of the Ad origin identified by in vitro studies are in fact important for viral DNA replication in infected cells. In addition, we found that two separate point mutations which lie outside these two sequence domains, and which have little or no effect upon DNA replication in vitro, also reduce the apparent efficiency of plasmid replication in vivo. Thus, there may be elements of the Ad DNA origin of replication which have not yet been identified by in vitro studies.

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Selected References

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

  1. Bellett A. J., Younghusband H. B. Replication of the DNA of chick embryo lethal orphan virus. J Mol Biol. 1972 Dec 30;72(3):691–709. doi: 10.1016/0022-2836(72)90185-4. [DOI] [PubMed] [Google Scholar]
  2. Carusi E. A. Evidence for blocked 5'-termini in human adenovirus DNA. Virology. 1977 Jan;76(1):380–394. doi: 10.1016/0042-6822(77)90310-5. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. 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]
  6. 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]
  7. Challberg M. D., Rawlins D. R. Template requirements for the initiation of adenovirus DNA replication. Proc Natl Acad Sci U S A. 1984 Jan;81(1):100–104. doi: 10.1073/pnas.81.1.100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Clewell D. B., Helinski D. R. Properties of a supercoiled deoxyribonucleic acid-protein relaxation complex and strand specificity of the relaxation event. Biochemistry. 1970 Oct 27;9(22):4428–4440. doi: 10.1021/bi00824a026. [DOI] [PubMed] [Google Scholar]
  9. Daniell E. Genome structure of incomplete particles of adenovirus. J Virol. 1976 Aug;19(2):685–708. doi: 10.1128/jvi.19.2.685-708.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Folger K. R., Wong E. A., Wahl G., Capecchi M. R. Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA molecules. Mol Cell Biol. 1982 Nov;2(11):1372–1387. doi: 10.1128/mcb.2.11.1372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Friefeld B. R., Krevolin M. D., Horwitz M. S. Effects of the adenovirus H5ts125 and H5ts107 DNA binding proteins on DNA replication in vitro. Virology. 1983 Jan 30;124(2):380–389. doi: 10.1016/0042-6822(83)90354-9. [DOI] [PubMed] [Google Scholar]
  13. Frost E., Williams J. Mapping temperature-sensitive and host-range mutations of adenovirus type 5 by marker rescue. Virology. 1978 Nov;91(1):39–50. doi: 10.1016/0042-6822(78)90353-7. [DOI] [PubMed] [Google Scholar]
  14. Garon C. F., Berry K. W., Rose J. A. A unique form of terminal redundancy in adenovirus DNA molecules. Proc Natl Acad Sci U S A. 1972 Sep;69(9):2391–2395. doi: 10.1073/pnas.69.9.2391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gelinas R. E., Myers P. A., Roberts R. J. Two sequence-specific endonucleases from Moraxella bovis. J Mol Biol. 1977 Jul;114(1):169–179. doi: 10.1016/0022-2836(77)90290-x. [DOI] [PubMed] [Google Scholar]
  16. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  17. Guggenheimer R. A., Nagata K., Lindenbaum J., Hurwitz J. Protein-primed replication of plasmids containing the terminus of the adenovirus genome. I. Characterization of an in vitro DNA replication system dependent on adenoviral DNA sequences. J Biol Chem. 1984 Jun 25;259(12):7807–7814. [PubMed] [Google Scholar]
  18. Guggenheimer R. A., Stillman B. W., Nagata K., Tamanoi F., Hurwitz J. DNA sequences required for the in vitro replication of adenovirus DNA. Proc Natl Acad Sci U S A. 1984 May;81(10):3069–3073. doi: 10.1073/pnas.81.10.3069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hanahan D., Gluzman Y. Rescue of functional replication origins from embedded configurations in a plasmid carrying the adenovirus genome. Mol Cell Biol. 1984 Feb;4(2):302–309. doi: 10.1128/mcb.4.2.302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hay R. T., Stow N. D., McDougall I. M. Replication of adenovirus mini-chromosomes. J Mol Biol. 1984 Jun 5;175(4):493–510. doi: 10.1016/0022-2836(84)90181-5. [DOI] [PubMed] [Google Scholar]
  21. Hay R. T. The origin of adenovirus DNA replication: minimal DNA sequence requirement in vivo. EMBO J. 1985 Feb;4(2):421–426. doi: 10.1002/j.1460-2075.1985.tb03645.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. doi: 10.1016/0022-2836(67)90307-5. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Kelly T. J., Jr, Lechner R. L. The structure of replicating adenovirus DNA molecules: characterization of DNA-protein complexes from infected cells. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 2):721–728. doi: 10.1101/sqb.1979.043.01.080. [DOI] [PubMed] [Google Scholar]
  25. Lally C., Dörper T., Gröger W., Antoine G., Winnacker E. L. A size analysis of the adenovirus replicon. EMBO J. 1984 Feb;3(2):333–337. doi: 10.1002/j.1460-2075.1984.tb01807.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Miller J. F., Malamy M. H. Identification of the pifC gene and its role in negative control of F factor pif gene expression. J Bacteriol. 1983 Oct;156(1):338–347. doi: 10.1128/jb.156.1.338-347.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. Nagata K., Guggenheimer R. A., Hurwitz J. Specific binding of a cellular DNA replication protein to the origin of replication of adenovirus DNA. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6177–6181. doi: 10.1073/pnas.80.20.6177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ostrove J. M., Rosenfeld P., Williams J., Kelly T. J., Jr In vitro complementation as an assay for purification of adenovirus DNA replication proteins. Proc Natl Acad Sci U S A. 1983 Feb;80(4):935–939. doi: 10.1073/pnas.80.4.935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Pearson G. D., Hanawalt P. C. Isolation of DNA replication complexes from uninfected and adenovirus-infected HeLa cells. J Mol Biol. 1971 Nov 28;62(1):65–80. doi: 10.1016/0022-2836(71)90131-8. [DOI] [PubMed] [Google Scholar]
  34. Peden K. W., Pipas J. M., Pearson-White S., Nathans D. Isolation of mutants of an animal virus in bacteria. Science. 1980 Sep 19;209(4463):1392–1396. doi: 10.1126/science.6251547. [DOI] [PubMed] [Google Scholar]
  35. Perucho M., Hanahan D., Lipsich L., Wigler M. Isolation of the chicken thymidine kinase gene by plasmid rescue. Nature. 1980 May 22;285(5762):207–210. doi: 10.1038/285207a0. [DOI] [PubMed] [Google Scholar]
  36. Pincus S., Robertson W., Rekosh D. Characterization of the effect of aphidicolin on adenovirus DNA replication: evidence in support of a protein primer model of initiation. Nucleic Acids Res. 1981 Oct 10;9(19):4919–4938. doi: 10.1093/nar/9.19.4919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rawlins D. R., Rosenfeld P. J., Wides R. J., Challberg M. D., Kelly T. J., Jr Structure and function of the adenovirus origin of replication. Cell. 1984 May;37(1):309–319. doi: 10.1016/0092-8674(84)90327-1. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Robins D. M., Ripley S., Henderson A. S., Axel R. Transforming DNA integrates into the host chromosome. Cell. 1981 Jan;23(1):29–39. doi: 10.1016/0092-8674(81)90267-1. [DOI] [PubMed] [Google Scholar]
  40. Shortle D., Nathans D. Local mutagenesis: a method for generating viral mutants with base substitutions in preselected regions of the viral genome. Proc Natl Acad Sci U S A. 1978 May;75(5):2170–2174. doi: 10.1073/pnas.75.5.2170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. 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]
  43. Stillman B. W., Bellett A. J. An adenovirus protein associated with the ends of replicating DNA molecules. Virology. 1979 Feb;93(1):69–79. doi: 10.1016/0042-6822(79)90276-9. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. Stillman B. W., Topp W. C., Engler J. A. Conserved sequences at the origin of adenovirus DNA replication. J Virol. 1982 Nov;44(2):530–537. doi: 10.1128/jvi.44.2.530-537.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Stow N. D. The infectivity of adenovirus genomes lacking DNA sequences from their left-hand termini. Nucleic Acids Res. 1982 Sep 11;10(17):5105–5119. doi: 10.1093/nar/10.17.5105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Tamanoi F., Stillman B. W. Function of adenovirus terminal protein in the initiation of DNA replication. Proc Natl Acad Sci U S A. 1982 Apr;79(7):2221–2225. doi: 10.1073/pnas.79.7.2221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tamanoi F., Stillman B. W. Initiation of adenovirus DNA replication in vitro requires a specific DNA sequence. Proc Natl Acad Sci U S A. 1983 Nov;80(21):6446–6450. doi: 10.1073/pnas.80.21.6446. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Tolun A., Aleström P., Pettersson U. Sequence of inverted terminal repetitions from different adenoviruses: demonstration of conserved sequences and homology between SA7 termini and SV40 DNA. Cell. 1979 Jul;17(3):705–713. doi: 10.1016/0092-8674(79)90277-0. [DOI] [PubMed] [Google Scholar]
  50. Wang K., Pearson G. D. Adenovirus sequences required for replication in vivo. Nucleic Acids Res. 1985 Jul 25;13(14):5173–5187. doi: 10.1093/nar/13.14.5173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wolfson J., Dressler D. Adenovirus-2 DNA contains an inverted terminal repetition. Proc Natl Acad Sci U S A. 1972 Oct;69(10):3054–3057. doi: 10.1073/pnas.69.10.3054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. van Bergen B. G., van der Ley P. A., van Driel W., van Mansfeld A. D., van der Vliet P. C. Replication of origin containing adenovirus DNA fragments that do not carry the terminal protein. Nucleic Acids Res. 1983 Apr 11;11(7):1975–1989. doi: 10.1093/nar/11.7.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. van Wielink P. S., Naaktgeboren N., Sussenbach J. S. Presence of protein at the termini of intracellular adenovirus type 5 DNA. Biochim Biophys Acta. 1979 Jun 20;563(1):89–99. doi: 10.1016/0005-2787(79)90010-8. [DOI] [PubMed] [Google Scholar]
  54. van der Eb A. J. Intermediates in type 5 adenovirus DNA replication. Virology. 1973 Jan;51(1):11–23. doi: 10.1016/0042-6822(73)90361-9. [DOI] [PubMed] [Google Scholar]
  55. van der Vliet P. C., Sussenbach J. S. The mechanism of adenovirus-DNA synthesis in isolated nuclei. Eur J Biochem. 1972 Nov 7;30(3):584–592. doi: 10.1111/j.1432-1033.1972.tb02130.x. [DOI] [PubMed] [Google Scholar]

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