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. 1991 Jul;65(7):3496–3503. doi: 10.1128/jvi.65.7.3496-3503.1991

The yeast GAL4 protein transactivates the polyomavirus origin of DNA replication in mouse cells.

M Baru 1, M Shlissel 1, H Manor 1
PMCID: PMC241338  PMID: 1645781

Abstract

We have replaced the polyomavirus (Py) enhancer, which is an essential component of the Py origin of DNA replication (ori), with five repeats of a 17-bp oligonucleotide including the yeast GAL4 upstream activating sequence (5xGAL4 sites). Plasmids containing this modified Py ori, designated test plasmids, and plasmids encoding either the GAL4 transcriptional activator protein or various derivatives of this protein were cotransfected into mouse cells which constitutively synthesize a temperature-sensitive Py large tumor antigen (T-Ag). Replication of the test plasmids was monitored by Southern blot determinations of the amounts of plasmid DNA that became resistant to cleavage by the enzyme DpnI. These studies showed that in the presence of a functional T-Ag, the GAL4 protein, and hybrid proteins including the GAL4 DNA-binding domain and the activating domain of the adenovirus E1a or herpesvirus VP16 protein transactivated the modified Py ori. A truncated protein including just the GAL4 DNA-binding domain was inactive in these assays. The authentic GAL4 protein was found to be a more efficient replication transactivator than the hybrid proteins. In contrast, chloramphenicol acetyltransferase assays showed that the hybrid proteins were more efficient transcriptional activators than the GAL4 protein. The extent of the GAL4-dependent replication of a plasmid in which the Py early promoter was deleted was 55% lower than that of a plasmid including the promoter. However, the extents of replication of plasmids including two tandem repeats of the remaining Py origin core and 5xGAL4 sites or two origin cores flanking a single cluster of 5xGAL4 sites were 4.8- and 1.6-fold higher than that of the plasmid including a single copy of each element. The replication of a plasmid including two clusters of 5xGAL4 sites flanking a single origin core was below the limit of detection of our assays. These results indicate that the GAL4 and hybrid transactivators do not activate the Py ori by virtue of their interactions with transcription factors that bind promoter elements. Rather, it appears that these activator proteins may interact with the replication initiation complexes, thereby facilitating or inhibiting the initiation of replication.

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Selected References

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

  1. Bennett E. R., Naujokas M., Hassell J. A. Requirements for species-specific papovavirus DNA replication. J Virol. 1989 Dec;63(12):5371–5385. doi: 10.1128/jvi.63.12.5371-5385.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brand A. H., Micklem G., Nasmyth K. A yeast silencer contains sequences that can promote autonomous plasmid replication and transcriptional activation. Cell. 1987 Dec 4;51(5):709–719. doi: 10.1016/0092-8674(87)90094-8. [DOI] [PubMed] [Google Scholar]
  3. Campbell B. A., Villarreal L. P. Functional analysis of the individual enhancer core sequences of polyomavirus: cell-specific uncoupling of DNA replication from transcription. Mol Cell Biol. 1988 May;8(5):1993–2004. doi: 10.1128/mcb.8.5.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cereghini S., Herbomel P., Jouanneau J., Saragosti S., Katinka M., Bourachot B., de Crombrugghe B., Yaniv M. Structure and function of the promoter-enhancer region of polyoma and SV40. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 2):935–944. doi: 10.1101/sqb.1983.047.01.107. [DOI] [PubMed] [Google Scholar]
  5. Cheng L., Kelly T. J. Transcriptional activator nuclear factor I stimulates the replication of SV40 minichromosomes in vivo and in vitro. Cell. 1989 Nov 3;59(3):541–551. doi: 10.1016/0092-8674(89)90037-8. [DOI] [PubMed] [Google Scholar]
  6. Cowie A., Kamen R. Multiple binding sites for polyomavirus large T antigen within regulatory sequences of polyomavirus DNA. J Virol. 1984 Dec;52(3):750–760. doi: 10.1128/jvi.52.3.750-760.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. DePamphilis M. L. Transcriptional elements as components of eukaryotic origins of DNA replication. Cell. 1988 Mar 11;52(5):635–638. doi: 10.1016/0092-8674(88)90398-4. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Guo Z. S., Gutierrez C., Heine U., Sogo J. M., Depamphilis M. L. Origin auxiliary sequences can facilitate initiation of simian virus 40 DNA replication in vitro as they do in vivo. Mol Cell Biol. 1989 Sep;9(9):3593–3602. doi: 10.1128/mcb.9.9.3593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gutierrez C., Guo Z. S., Roberts J., DePamphilis M. L. Simian virus 40 origin auxiliary sequences weakly facilitate T-antigen binding but strongly facilitate DNA unwinding. Mol Cell Biol. 1990 Apr;10(4):1719–1728. doi: 10.1128/mcb.10.4.1719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hendrickson E. A., Fritze C. E., Folk W. R., DePamphilis M. L. The origin of bidirectional DNA replication in polyoma virus. EMBO J. 1987 Jul;6(7):2011–2018. doi: 10.1002/j.1460-2075.1987.tb02465.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Hodgson C. P., Fisk R. Z. Hybridization probe size control: optimized 'oligolabelling'. Nucleic Acids Res. 1987 Aug 11;15(15):6295–6295. doi: 10.1093/nar/15.15.6295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Johnson P. F., McKnight S. L. Eukaryotic transcriptional regulatory proteins. Annu Rev Biochem. 1989;58:799–839. doi: 10.1146/annurev.bi.58.070189.004055. [DOI] [PubMed] [Google Scholar]
  15. Jones K. A., Kadonaga J. T., Rosenfeld P. J., Kelly T. J., Tjian R. A cellular DNA-binding protein that activates eukaryotic transcription and DNA replication. Cell. 1987 Jan 16;48(1):79–89. doi: 10.1016/0092-8674(87)90358-8. [DOI] [PubMed] [Google Scholar]
  16. Jones N. C., Rigby P. W., Ziff E. B. Trans-acting protein factors and the regulation of eukaryotic transcription: lessons from studies on DNA tumor viruses. Genes Dev. 1988 Mar;2(3):267–281. doi: 10.1101/gad.2.3.267. [DOI] [PubMed] [Google Scholar]
  17. Kakidani H., Ptashne M. GAL4 activates gene expression in mammalian cells. Cell. 1988 Jan 29;52(2):161–167. doi: 10.1016/0092-8674(88)90504-1. [DOI] [PubMed] [Google Scholar]
  18. Katinka M., Yaniv M. DNA replication origin of polyoma virus: early proximal boundary. J Virol. 1983 Jul;47(1):244–248. doi: 10.1128/jvi.47.1.244-248.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kern F. G., Basilico C. An inducible eukaryotic host-vector expression system: amplification of genes under the control of the polyoma late promoter in a cell line producing a thermolabile large T antigen. Gene. 1986;43(3):237–245. doi: 10.1016/0378-1119(86)90212-x. [DOI] [PubMed] [Google Scholar]
  20. Kimmerly W., Buchman A., Kornberg R., Rine J. Roles of two DNA-binding factors in replication, segregation and transcriptional repression mediated by a yeast silencer. EMBO J. 1988 Jul;7(7):2241–2253. doi: 10.1002/j.1460-2075.1988.tb03064.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lillie J. W., Green M. R. Transcription activation by the adenovirus E1a protein. Nature. 1989 Mar 2;338(6210):39–44. doi: 10.1038/338039a0. [DOI] [PubMed] [Google Scholar]
  22. Luthman H., Nilsson M. G., Magnusson G. Non-contiguous segments of the polyoma genome required in cis for DNA replication. J Mol Biol. 1982 Nov 15;161(4):533–550. doi: 10.1016/0022-2836(82)90406-5. [DOI] [PubMed] [Google Scholar]
  23. Ma J., Ptashne M. Deletion analysis of GAL4 defines two transcriptional activating segments. Cell. 1987 Mar 13;48(5):847–853. doi: 10.1016/0092-8674(87)90081-x. [DOI] [PubMed] [Google Scholar]
  24. Maniatis T., Goodbourn S., Fischer J. A. Regulation of inducible and tissue-specific gene expression. Science. 1987 Jun 5;236(4806):1237–1245. doi: 10.1126/science.3296191. [DOI] [PubMed] [Google Scholar]
  25. Manor H., Neer A. Effects of cycloheximide on virus RNA replication in an inducible line of polyoma-transformed rat cells. Cell. 1975 Jul;5(3):311–318. doi: 10.1016/0092-8674(75)90106-3. [DOI] [PubMed] [Google Scholar]
  26. McKnight S., Tjian R. Transcriptional selectivity of viral genes in mammalian cells. Cell. 1986 Sep 12;46(6):795–805. doi: 10.1016/0092-8674(86)90061-9. [DOI] [PubMed] [Google Scholar]
  27. Mendelsohn E., Baran N., Neer A., Manor H. Integration site of polyoma virus DNA in the inducible LPT line of polyoma-transformed rat cells. J Virol. 1982 Jan;41(1):192–209. doi: 10.1128/jvi.41.1.192-209.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mermod N., O'Neill E. A., Kelly T. J., Tjian R. The proline-rich transcriptional activator of CTF/NF-I is distinct from the replication and DNA binding domain. Cell. 1989 Aug 25;58(4):741–753. doi: 10.1016/0092-8674(89)90108-6. [DOI] [PubMed] [Google Scholar]
  29. O'Neill E. A., Fletcher C., Burrow C. R., Heintz N., Roeder R. G., Kelly T. J. Transcription factor OTF-1 is functionally identical to the DNA replication factor NF-III. Science. 1988 Sep 2;241(4870):1210–1213. doi: 10.1126/science.3413485. [DOI] [PubMed] [Google Scholar]
  30. Passmore S., Maine G. T., Elble R., Christ C., Tye B. K. Saccharomyces cerevisiae protein involved in plasmid maintenance is necessary for mating of MAT alpha cells. J Mol Biol. 1988 Dec 5;204(3):593–606. doi: 10.1016/0022-2836(88)90358-0. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Pomerantz B. J., Mueller C. R., Hassell J. A. Polyomavirus large T antigen binds independently to multiple, unique regions on the viral genome. J Virol. 1983 Sep;47(3):600–610. doi: 10.1128/jvi.47.3.600-610.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ptashne M. How eukaryotic transcriptional activators work. Nature. 1988 Oct 20;335(6192):683–689. doi: 10.1038/335683a0. [DOI] [PubMed] [Google Scholar]
  34. Rochford R., Davis C. T., Yoshimoto K. K., Villarreal L. P. Minimal subenhancer requirements for high-level polyomavirus DNA replication: a cell-specific synergy of PEA3 and PEA1 sites. Mol Cell Biol. 1990 Sep;10(9):4996–5001. doi: 10.1128/mcb.10.9.4996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sadowski I., Ma J., Triezenberg S., Ptashne M. GAL4-VP16 is an unusually potent transcriptional activator. Nature. 1988 Oct 6;335(6190):563–564. doi: 10.1038/335563a0. [DOI] [PubMed] [Google Scholar]
  36. Seed B., Sheen J. Y. A simple phase-extraction assay for chloramphenicol acyltransferase activity. Gene. 1988 Jul 30;67(2):271–277. doi: 10.1016/0378-1119(88)90403-9. [DOI] [PubMed] [Google Scholar]
  37. Tang W. J., Berger S. L., Triezenberg S. J., Folk W. R. Nucleotides in the polyomavirus enhancer that control viral transcription and DNA replication. Mol Cell Biol. 1987 May;7(5):1681–1690. doi: 10.1128/mcb.7.5.1681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Triezenberg S. J., Folk W. R. Essential nucleotides in the polyomavirus origin region. J Virol. 1984 Aug;51(2):437–444. doi: 10.1128/jvi.51.2.437-444.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Veldman G. M., Lupton S., Kamen R. Polyomavirus enhancer contains multiple redundant sequence elements that activate both DNA replication and gene expression. Mol Cell Biol. 1985 Apr;5(4):649–658. doi: 10.1128/mcb.5.4.649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Verrijzer C. P., Kal A. J., Van der Vliet P. C. The DNA binding domain (POU domain) of transcription factor oct-1 suffices for stimulation of DNA replication. EMBO J. 1990 Jun;9(6):1883–1888. doi: 10.1002/j.1460-2075.1990.tb08314.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  42. Webster N., Jin J. R., Green S., Hollis M., Chambon P. The yeast UASG is a transcriptional enhancer in human HeLa cells in the presence of the GAL4 trans-activator. Cell. 1988 Jan 29;52(2):169–178. doi: 10.1016/0092-8674(88)90505-3. [DOI] [PubMed] [Google Scholar]
  43. Zhu Z. Y., Veldman G. M., Cowie A., Carr A., Schaffhausen B., Kamen R. Construction and functional characterization of polyomavirus genomes that separately encode the three early proteins. J Virol. 1984 Jul;51(1):170–180. doi: 10.1128/jvi.51.1.170-180.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. de Villiers J., Schaffner W., Tyndall C., Lupton S., Kamen R. Polyoma virus DNA replication requires an enhancer. Nature. 1984 Nov 15;312(5991):242–246. doi: 10.1038/312242a0. [DOI] [PubMed] [Google Scholar]

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