Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

DNA replication control through interaction of E2F–RB and the origin recognition complex

Abstract

The E2F transcription factor and retinoblastoma protein control cell-cycle progression and DNA replication during S phase. Mutations in the Drosophila dE2F1 and dDP genes affect the origin recognition complex (DmORC) and initiation of replication at the chorion gene replication origin. Here we show that mutants of Rbf (an retinoblastoma protein homologue) fail to limit DNA replication. We also show that the dDP, dE2F1 and Rbf proteins are located in a complex with DmORC, and that dE2F1 and DmORC are bound to the chorion origin of replication in vivo. Our results indicate that dE2F1 and Rbf function together at replication origins to limit DNA replication through interactions with DmORC.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Follicle cells of Rbf female-sterile mutants have DNA replication and amplification defects.
Figure 2: dE2F1 interacts with DmORC and binds to origin DNA.
Figure 3: A stable and functional truncated dE2F1 protein does not interact with Rbf or DmORC.
Figure 4: Model for the dE2F–Rbf regulation of endo cycles and DmORC activity during gene amplification.

Similar content being viewed by others

References

  1. Royzman, I. & Orr-Weaver, T. L. S phase and differential DNA replication during Drosophila oogenesis. Genes Cells 3, 767–776 (1998).

    Article  CAS  Google Scholar 

  2. Calvi, B. R. & Spradling, A. C. in Genetic Approaches to Eukaryotic Replication and Repair (ed. Fisher, P.) 407–417 (Academic, New York, 1999).

    Google Scholar 

  3. Dutta, A. & Bell, S. P. Initiation of DNA replication in eukaryotic cells. Annu. Rev. Cell Dev. Biol. 13, 293–332 (1997).

    Article  CAS  Google Scholar 

  4. Royzman, I., Austin, R. J., Bosco, G., Bell, S. P. & Orr-Weaver, T. L. ORC localization in Drosophila follicle cells and the effects of mutations in dE2F and dDP. Genes Dev. 13, 827–840 ( 1999).

    Article  CAS  Google Scholar 

  5. Asano, M. & Wharton, R. P. E2F mediates developmental and cell cycle regulation of ORC1 in Drosophila. EMBO J. 18, 2435–2448 (1999).

    Article  CAS  Google Scholar 

  6. Austin, R. J., Orr-Weaver, T. L. & Bell, S. P. Drosophila ORC specifically binds to ACE3 , an origin of DNA replication control element. Genes Dev. 13, 2639–2649 ( 1999).

    Article  CAS  Google Scholar 

  7. Chesnokov, I., Gossen, M., Remus, D. & Botchan, M. Assembly of functionally active Drosophila origin recognition complex from recombinant proteins . Genes Dev. 13, 1289–1296 (1999).

    Article  CAS  Google Scholar 

  8. Spradling, A. C. ORC binding, gene amplification, and the nature of metazoan replication origins . Genes Dev. 13, 2619–2623 (1999).

    Article  CAS  Google Scholar 

  9. Dyson, N. The regulation of E2F by pRB-family proteins. Genes Dev. 12, 2245–2262 (1998).

    Article  CAS  Google Scholar 

  10. Weintraub, S. J., Prater, C. A. & Dean, D. C. Retinoblastoma protein switches the E2F site from positive to negative element. Nature 358, 259–261 (1992).

    Article  CAS  Google Scholar 

  11. Weinberg, R. A. The retinoblastoma protein and cell cycle control. Cell 81, 323–330 (1995).

    Article  CAS  Google Scholar 

  12. Helin, K. et al. Heterodimerization of the transcription factors E2F-1 and DP-1 leads to cooperative trans-activation. Genes Dev. 7, 1850–1861 (1993).

    Article  CAS  Google Scholar 

  13. Brehm, A. et al. Retinoblastoma protein recruits histone deacetylase to repress transcription. Nature 391, 597– 601 (1998).

    Article  CAS  Google Scholar 

  14. Luo, R. X., Postigo, A. A. & Dean, D. C. Rb interacts with histone deacetylase to repress transcription. Cell 92, 463– 473 (1998).

    Article  CAS  Google Scholar 

  15. Magnaghi-Jaulin, L. et al. Retinoblastoma protein represses transcription by recruiting a histone deacetylase. Nature 391, 601– 605 (1998).

    Article  CAS  Google Scholar 

  16. Krek, W., Xu, G. & Livingston, D. M. Cyclin A-kinase regulation of E2F-1 DNA binding function underlies suppression of an S phase checkpoint. Cell 83, 1149–1158 (1995).

    Article  CAS  Google Scholar 

  17. Chew, Y. P., Ellis, M., Wilkie, S. & Mittnacht, S. pRB phosphorylation mutants reveal role of pRB in regulating S phase completion by a mechanism independent of E2F. Oncogene 17, 2177– 2186 (1998).

    Article  CAS  Google Scholar 

  18. Knudsen, K. E. et al. RB-Dependent S-phase response to DNA damage. Mol. Cell. Biol. 20, 7751–7763 (2000).

    Article  CAS  Google Scholar 

  19. Sterner, J. M., Dew-Knight, S., Musahl, C., Kornbluth, S. & Horowitz, J. M. Negative regulation of DNA replication by the retinoblastoma protein is mediated by its association with MCM7. Mol. Cell. Biol. 18, 2748–2757 (1998).

    Article  CAS  Google Scholar 

  20. Sawado, T. et al. dE2F2, a novel E2F-family transcription factor in Drosophila melanogaster. Biochem. Biophys. Res. Commun. 251 , 409–415 (1998).

    Article  CAS  Google Scholar 

  21. Ohtani, K. & Nevins, J. R. Functional properties of a Drosophila homolog of the E2F1 gene. Mol. Cell. Biol. 14, 1603–1612 (1994).

    Article  CAS  Google Scholar 

  22. Royzman, I., Whittaker, A. J. & Orr-Weaver, T. L. Mutations in Drosophila DP and E2F distinguish G1-S progression from an associated transcriptional program. Genes Dev. 11, 1999–2011 (1997).

    Article  CAS  Google Scholar 

  23. Duronio, R. J., Bonnette, P. C. & O'Farrell, P. H. Mutations of the Drosophila dDP, dE2F, and cyclin E genes reveal distinct roles for the E2F-DP transcription factor and cyclin E during the G1-S transition. Mol. Cell. Biol. 18, 141–151 (1998).

    Article  CAS  Google Scholar 

  24. Du, W. Suppression of the rbf null mutants by a de2f1 allele that lacks transactivation domain . Development 127, 367– 379 (2000).

    CAS  PubMed  Google Scholar 

  25. Du, W. & Dyson, N. The role of RBF in the introduction of G1 regulation during Drosophila embryogenesis. EMBO J. 18, 916–925 ( 1999).

    Article  CAS  Google Scholar 

  26. Calvi, B. R., Lilly, M. A. & Spradling, A. C. Cell cycle control of chorion gene amplification . Genes Dev. 12, 734–744 (1998).

    Article  CAS  Google Scholar 

  27. Lilly, M. A. & Spradling, A. C. The Drosophila endocycle is controlled by Cyclin E and lacks a checkpoint ensuring S-phase completion . Genes Dev. 10, 2514–2526 (1996).

    Article  CAS  Google Scholar 

  28. Lai, J. S. & Herr, W. Ethidium bromide provides a simple tool for identifying genuine DNA-independent protein associations. Proc. Natl Acad. Sci. USA 89, 6958– 6962 (1992).

    Article  CAS  Google Scholar 

  29. Strahl-Bolsinger, S., Hecht, A., Luo, K. & Grunstein, M. SIR2 and SIR4 interactions differ in core and extended telomeric heterochromatin in yeast . Genes Dev. 11, 83–93 (1997).

    Article  CAS  Google Scholar 

  30. Yamaguchi, M., Hayashi, Y., Hirose, F., Nishimoto, Y. & Matsukage, A. Distinct roles of E2F recognition sites as positive or negative elements in regulation of the DNA polymerase alpha 180 kDa catalytic subunit gene promoter during Drosophila development. Nucleic Acids Res 25, 3847–3854 (1997).

    Article  CAS  Google Scholar 

  31. Zhang, H. S., Postigo, A. A. & Dean, D. C. Active transcriptional repression by the Rb-E2F complex mediates G1 arrest. Cell 97, 53– 61 (1999).

    Article  CAS  Google Scholar 

  32. Sasaki, T., Sawado, T., Yamaguchi, M. & Shinomiya, T. Specification of regions of DNA replication initiation during embryogenesis in the 65-kilobase DNApolα-dE2F locus of Drosophila melanogaster . Mol. Cell. Biol. 19, 547– 555 (1999).

    Article  CAS  Google Scholar 

  33. Fenger, D. D. et al. PAN GU: a protein kinase that inhibits S phase and promotes mitosis in early Drosophila development. Development 127, 4763–4774 (2000).

    CAS  PubMed  Google Scholar 

  34. Asano, M., Nevins, J. R. & Wharton, R. P. Ectopic E2F expression induces S phase and apoptosis in Drosophila imaginal discs. Genes Dev. 10, 1422–1432 (1996).

    Article  CAS  Google Scholar 

  35. Elfring, L. K. et al. The Drosophila PLUTONIUM protein is a specialized cell cycle regulator required at the onset of development. Mol. Biol. Cell 8, 583–593 ( 1997).

    Article  CAS  Google Scholar 

  36. Du, W., Vidal, M., Xie, J.-E. & Dyson, N. RBF, a novel RB-related gene that regulates E2F activity and interacts with cyclin E in Drosophila. Genes Dev. 10, 1206–1218 (1996).

    Article  CAS  Google Scholar 

  37. Kennedy, B. K., Barbie, D. A., Classon, M., Dyson, N. & Harlow, E. Nuclear organisation of DNA replication in primary mammalian cells. Genes Dev. 14, 2855–2868 (2000).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We are grateful to S. Bell, R. J. Austin, M. Asano, R. Wharton and N. Dyson for providing antibodies, and H. Kashevsky for technical support in making antibodies. We thank B. Kennedy and E. Harlow for communicating results before publication. We thank J. Lees, R. Austin and S. Bell for many helpful discussions and comments on the manuscript. R. Weinberg, I. Rebay and S. Stewart also provided critical suggestions on the manuscript. The microscopy was done in the Keck Imaging Center at the Whitehead Institute. G.B. is a postdoctoral fellow of the Damon Runyon-Walter Winchell Fund. This work was supported by a grant from the American Heart Association and the NIH to T.O.-W. and a Margaret and Herman Solcol fellow of the Whitehead Institute and an NIH grant to W.D.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Terry L. Orr-Weaver.

Supplementary information

Download plugins

Figure S1. Quantification of chorion gene amplification in Rbf 120a /Rbf 14 -mutant follicle cells. Quantitative PCR was used to measure the levels of ACE3 and actin-gene DNA in egg-chambers at stage 1–8 (a) and at stage 13 (b) for Rbf120a/Rbf14 mutants and rbf14/+ sibling controls. For each sample three serial dilutions are shown from right to left, and the positions of the ACE3 and actin PCR fragments are indicated on the right. The actin gene is not amplified and serves as an internal standard for the amount of DNA in each sample. The level of amplification was measured by comparing the ratio of ACE3 to actin in stage 13 egg-chambers, which had undergone amplification, to the ratio of ACE3 to actin in stage 1–8 egg-chambers, which had not initiated amplification. Rbf120a/Rbf14-mutant follicle cells overamplify the chorion region relative to rbf14/ + sibling controls. (PDF 331 kb)

Figure S2. In vitro culture of egg-chambers with a-amanitin blocks reaper transcription but not the DNA-replication defect of Rbf120a/Rbf14 follicle cells. Egg-chambers were cultured in vitro for up to 6 h with or without α-amanitin. a, The reaper gene is induced in follicle cells of egg-chambers at stages 9 and 10 and was used to determine whether a-amanitin blocks transcription, as detected by in situ hybridization. b, Despite the effect of a-amanitin on reaper transcription, BrdU labelling showed that increased amplification and DNA replication still occur in Rbf120a/Rbf14 follicle cells.

Figure S3. Levels of truncated dE2F1i2 relative to full-length dE2F1 in different extracts. The ratios of dE2F1i2 (arrowhead) and dE2F1 (arrow) are shown for four different extracts (andash;d) on immunoblots of dE2F1 immunoprecipitations. In a, immunoprecipations of the same extract with anti-dDP and anti-dE2F1 antibodies are shown. This demonstrates that the reduced amount of dE2F1i2 relative to dE2F1 in the dDP immunoprecipitation reflects the amount of dE2F1i2 in the extract, rather than a weaker affinity for dDP.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bosco, G., Du, W. & Orr-Weaver, T. DNA replication control through interaction of E2F–RB and the origin recognition complex. Nat Cell Biol 3, 289–295 (2001). https://doi.org/10.1038/35060086

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/35060086

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing