Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1998 Mar 16;17(6):1810–1818. doi: 10.1093/emboj/17.6.1810

Transformation abrogates an early G1-phase arrest point required for specification of the Chinese hamster DHFR replication origin.

J R Wu 1, S M Keezer 1, D M Gilbert 1
PMCID: PMC1170528  PMID: 9501102

Abstract

The origin decision point (ODP) was originally identified as a distinct point during G1-phase when Chinese hamster ovary (CHO) cell nuclei experience a transition that is required for specific recognition of the dihydrofolate reductase (DHFR) origin locus by Xenopus egg extracts. Passage of cells through the ODP requires a mitogen-independent protein kinase that is activated prior to restriction point control. Here we show that inhibition of an early G1-phase protein kinase pathway by the addition of 2-aminopurine (2-AP) prior to the ODP arrests CHO cells in G1-phase. Transformation with simian virus 40 (SV40) abrogated this arrest point, resulting in the entry of cultured cells into S-phase in the presence of 2-AP and a disruption of the normal pattern of initiation sites at the DHFR locus. Cells treated with 2-AP after the ODP initiated replication specifically within the DHFR origin locus. Transient exposure of transformed cells to 2-AP during the ODP transition also disrupted origin choice, whereas non-transformed cells arrested in G1-phase and then passed through a delayed ODP after removal of 2-AP from the medium. We conclude that mammalian cells have many potential sites at which they can initiate replication. Normally, events occurring during the early G1-phase ODP transition determine which of these sites will be the preferred initiation site. However, if chromatin is exposed to S-phase-promoting factors prior to this transition, mammalian cells, like Xenopus and Drosophila embryos, can initiate replication without origin specification.

Full Text

The Full Text of this article is available as a PDF (451.5 KB).

Selected References

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

  1. Anderson S., DePamphilis M. L. Metabolism of Okazaki fragments during simian virus 40 DNA replication. J Biol Chem. 1979 Nov 25;254(22):11495–11504. [PubMed] [Google Scholar]
  2. Burhans W. C., Vassilev L. T., Caddle M. S., Heintz N. H., DePamphilis M. L. Identification of an origin of bidirectional DNA replication in mammalian chromosomes. Cell. 1990 Sep 7;62(5):955–965. doi: 10.1016/0092-8674(90)90270-o. [DOI] [PubMed] [Google Scholar]
  3. Burhans W. C., Vassilev L. T., Wu J., Sogo J. M., Nallaseth F. S., DePamphilis M. L. Emetine allows identification of origins of mammalian DNA replication by imbalanced DNA synthesis, not through conservative nucleosome segregation. EMBO J. 1991 Dec;10(13):4351–4360. doi: 10.1002/j.1460-2075.1991.tb05013.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Coverley D., Laskey R. A. Regulation of eukaryotic DNA replication. Annu Rev Biochem. 1994;63:745–776. doi: 10.1146/annurev.bi.63.070194.003525. [DOI] [PubMed] [Google Scholar]
  5. Crissman H. A., Gadbois D. M., Tobey R. A., Bradbury E. M. Transformed mammalian cells are deficient in kinase-mediated control of progression through the G1 phase of the cell cycle. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7580–7584. doi: 10.1073/pnas.88.17.7580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. DePamphilis M. L. Origins of DNA replication in metazoan chromosomes. J Biol Chem. 1993 Jan 5;268(1):1–4. [PubMed] [Google Scholar]
  7. Dijkwel P. A., Hamlin J. L. The Chinese hamster dihydrofolate reductase origin consists of multiple potential nascent-strand start sites. Mol Cell Biol. 1995 Jun;15(6):3023–3031. doi: 10.1128/mcb.15.6.3023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Distel B., Erdmann R., Gould S. J., Blobel G., Crane D. I., Cregg J. M., Dodt G., Fujiki Y., Goodman J. M., Just W. W. A unified nomenclature for peroxisome biogenesis factors. J Cell Biol. 1996 Oct;135(1):1–3. doi: 10.1083/jcb.135.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gadbois D. M., Crissman H. A., Tobey R. A., Bradbury E. M. Multiple kinase arrest points in the G1 phase of nontransformed mammalian cells are absent in transformed cells. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8626–8630. doi: 10.1073/pnas.89.18.8626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gilbert D. M., Cohen S. N. Bovine papilloma virus plasmids replicate randomly in mouse fibroblasts throughout S phase of the cell cycle. Cell. 1987 Jul 3;50(1):59–68. doi: 10.1016/0092-8674(87)90662-3. [DOI] [PubMed] [Google Scholar]
  11. Gilbert D. M., Cohen S. N. Position effects on the timing of replication of chromosomally integrated simian virus 40 molecules in Chinese hamster cells. Mol Cell Biol. 1990 Aug;10(8):4345–4355. doi: 10.1128/mcb.10.8.4345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gilbert D. M., Miyazawa H., DePamphilis M. L. Site-specific initiation of DNA replication in Xenopus egg extract requires nuclear structure. Mol Cell Biol. 1995 Jun;15(6):2942–2954. doi: 10.1128/mcb.15.6.2942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gruss C., Wu J., Koller T., Sogo J. M. Disruption of the nucleosomes at the replication fork. EMBO J. 1993 Dec;12(12):4533–4545. doi: 10.1002/j.1460-2075.1993.tb06142.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hamlin J. L., Dijkwel P. A. On the nature of replication origins in higher eukaryotes. Curr Opin Genet Dev. 1995 Apr;5(2):153–161. doi: 10.1016/0959-437x(95)80002-6. [DOI] [PubMed] [Google Scholar]
  15. Hamlin J. L., Mosca P. J., Levenson V. V. Defining origins of replication in mammalian cells. Biochim Biophys Acta. 1994 Dec 30;1198(2-3):85–111. doi: 10.1016/0304-419x(94)90008-6. [DOI] [PubMed] [Google Scholar]
  16. Hyrien O., Maric C., Méchali M. Transition in specification of embryonic metazoan DNA replication origins. Science. 1995 Nov 10;270(5238):994–997. doi: 10.1126/science.270.5238.994. [DOI] [PubMed] [Google Scholar]
  17. Hyrien O., Méchali M. Chromosomal replication initiates and terminates at random sequences but at regular intervals in the ribosomal DNA of Xenopus early embryos. EMBO J. 1993 Dec;12(12):4511–4520. doi: 10.1002/j.1460-2075.1993.tb06140.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Joo W. S., Luo X., Denis D., Kim H. Y., Rainey G. J., Jones C., Sreekumar K. R., Bullock P. A. Purification of the simian virus 40 (SV40) T antigen DNA-binding domain and characterization of its interactions with the SV40 origin. J Virol. 1997 May;71(5):3972–3985. doi: 10.1128/jvi.71.5.3972-3985.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Leno G. H., Downes C. S., Laskey R. A. The nuclear membrane prevents replication of human G2 nuclei but not G1 nuclei in Xenopus egg extract. Cell. 1992 Apr 3;69(1):151–158. doi: 10.1016/0092-8674(92)90126-w. [DOI] [PubMed] [Google Scholar]
  20. Little R. D., Platt T. H., Schildkraut C. L. Initiation and termination of DNA replication in human rRNA genes. Mol Cell Biol. 1993 Oct;13(10):6600–6613. doi: 10.1128/mcb.13.10.6600. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Little R. D., Schildkraut C. L. Initiation of latent DNA replication in the Epstein-Barr virus genome can occur at sites other than the genetically defined origin. Mol Cell Biol. 1995 May;15(5):2893–2903. doi: 10.1128/mcb.15.5.2893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ludlow J. W., DeCaprio J. A., Huang C. M., Lee W. H., Paucha E., Livingston D. M. SV40 large T antigen binds preferentially to an underphosphorylated member of the retinoblastoma susceptibility gene product family. Cell. 1989 Jan 13;56(1):57–65. doi: 10.1016/0092-8674(89)90983-5. [DOI] [PubMed] [Google Scholar]
  23. O'Keefe R. T., Henderson S. C., Spector D. L. Dynamic organization of DNA replication in mammalian cell nuclei: spatially and temporally defined replication of chromosome-specific alpha-satellite DNA sequences. J Cell Biol. 1992 Mar;116(5):1095–1110. doi: 10.1083/jcb.116.5.1095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pelizon C., Diviacco S., Falaschi A., Giacca M. High-resolution mapping of the origin of DNA replication in the hamster dihydrofolate reductase gene domain by competitive PCR. Mol Cell Biol. 1996 Oct;16(10):5358–5364. doi: 10.1128/mcb.16.10.5358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Shinomiya T., Ina S. DNA replication of histone gene repeats in Drosophila melanogaster tissue culture cells: multiple initiation sites and replication pause sites. Mol Cell Biol. 1993 Jul;13(7):4098–4106. doi: 10.1128/mcb.13.7.4098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Shinomiya T., Ina S. Mapping an initiation region of DNA replication at a single-copy chromosomal locus in Drosophila melanogaster cells by two-dimensional gel methods and PCR-mediated nascent-strand analysis: multiple replication origins in a broad zone. Mol Cell Biol. 1994 Nov;14(11):7394–7403. doi: 10.1128/mcb.14.11.7394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wu J. R., Gilbert D. M. A distinct G1 step required to specify the Chinese hamster DHFR replication origin. Science. 1996 Mar 1;271(5253):1270–1272. doi: 10.1126/science.271.5253.1270. [DOI] [PubMed] [Google Scholar]
  28. Wu J. R., Gilbert D. M. The replication origin decision point is a mitogen-independent, 2-aminopurine-sensitive, G1-phase event that precedes restriction point control. Mol Cell Biol. 1997 Aug;17(8):4312–4321. doi: 10.1128/mcb.17.8.4312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wu J. R., Yu G., Gilbert D. M. Origin-specific initiation of mammalian nuclear DNA replication in a Xenopus cell-free system. Methods. 1997 Nov;13(3):313–324. doi: 10.1006/meth.1997.0530. [DOI] [PubMed] [Google Scholar]
  30. Zetterberg A., Larsson O., Wiman K. G. What is the restriction point? Curr Opin Cell Biol. 1995 Dec;7(6):835–842. doi: 10.1016/0955-0674(95)80067-0. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES