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The DNA replication checkpoint response stabilizes stalled replication forks

Nature volume 412, pages 557–561 (2001)Cite this article

Abstract

In response to DNA damage and blocks to replication, eukaryotes activate the checkpoint pathways that prevent genomic instability and cancer by coordinating cell cycle progression with DNA repair1,2,3,4,5. In budding yeast, the checkpoint response requires the Mec1-dependent activation of the Rad53 protein kinase3,4,6. Active Rad53 slows DNA synthesis when DNA is damaged7 and prevents firing of late origins of replication8,9. Further, rad53 mutants are unable to recover from a replication block10. Mec1 and Rad53 also modulate the phosphorylation state of different DNA replication and repair enzymes6,11,12,13. Little is known of the mechanisms by which checkpoint pathways interact with the replication apparatus when DNA is damaged or replication blocked. We used the two-dimensional gel technique14 to examine replication intermediates in response to hydroxyurea-induced replication blocks. Here we show that hydroxyurea-treated rad53 mutants accumulate unusual DNA structures at replication forks. The persistence of these abnormal molecules during recovery from the hydroxyurea block correlates with the inability to dephosphorylate Rad53. Further, Rad53 is required to properly maintain stable replication forks during the block. We propose that Rad53 prevents collapse of the fork when replication pauses.

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Figure 1: rad53-K227A mutant cells accumulate abnormal DNA structures at ARS305 in response to hydroxyurea treatment.
Figure 2: Analysis of replication intermediates in chromosome III regions adjacent to the ARS305 origin.
Figure 3: Y-shaped molecules in rad53-K227A mutant cells persist after hydroxyurea removal.
Figure 4: Proper maintenance of hydroxyurea-arrested replication forks requires Rad53 activity.

References

  1. Elledge, S. J. Cell cycle checkpoints: preventing an identity crisis. Science 274, 1664–1672 (1996).

    Article  ADS  CAS  Google Scholar 

  2. Weinert, T. DNA damage checkpoints update: getting molecular. Curr. Opin. Genet. Dev. 8, 185–193 (1998).

    Article  CAS  Google Scholar 

  3. Lowndes, N. F. & Murguia, J. R. Sensing and responding to DNA damage. Curr. Opin. Genet. Dev. 10, 17–25 (2000).

    Article  CAS  Google Scholar 

  4. Foiani, M. et al. DNA damage checkpoints and DNA replication controls in Saccharomyces cerevisiae. Mutat. Res. 21, 286–294 (2000).

    Google Scholar 

  5. Weinert, T. Yeast checkpoints controls and relevance to cancer. Cancer Surv. 29, 109–132 (1997).

    CAS  PubMed  Google Scholar 

  6. Pellicioli, A. et al. Activation of Rad53 kinase in response to DNA damage and its effect in modulating phosphorylation of the lagging strand DNA polymerase. EMBO J. 18, 6561–6572 (1999).

    Article  CAS  Google Scholar 

  7. Paulovich, A. G. & Hartwell, L. H. A checkpoint regulates the rate of progression through S-phase in S. cerevisiae in response to DNA damage. Cell 82, 841–847 (1995).

    Article  CAS  Google Scholar 

  8. Santocanale, C. & Diffley, J. F. X. A Mec1- and Rad53-dependent checkpoint controls late-firing origins of DNA replication. Nature 395, 615–618 (1998).

    Article  ADS  CAS  Google Scholar 

  9. Shirahige, K. et al. Regulation of DNA-replication origins during cell-cycle progression. Nature 395, 618–621 (1998).

    Article  ADS  CAS  Google Scholar 

  10. Desany, B. A., Alcasabas, A. A., Bachant, J. B. & Elledge, S. J. Recovery from DNA replicational stress is the essential function of the S-phase checkpoint pathway. Genes Dev. 12, 2956–2970 (1998).

    Article  CAS  Google Scholar 

  11. Brush, G., Morrow, D. M., Heiter, P. & Kelly, T. J. The ATM homolog MEC1 is required for phosphorylation of replication protein A in yeast. Proc. Natl Acad. Sci. USA 93, 15075–15080 (1996).

    Article  ADS  CAS  Google Scholar 

  12. Liberi, G. et al. Srs2 DNA helicase is involved in checkpoint response and its regulation requires a functional Mec1-dependent pathway and CDK1 activity. EMBO J. 19, 5027–5038 (2000).

    Article  CAS  Google Scholar 

  13. Bashkirov, V. I. et al. DNA repair protein Rad55 is a terminal substrate of the DNA damage checkpoints. Mol. Cell. Biol. 20, 4393–4404 (2000).

    Article  CAS  Google Scholar 

  14. Brewer, B. J. & Fangman, W. L. The localization of replication origins on ARS plasmids in S. cerevisiae. Cell 51, 463–471 (1987).

    Article  CAS  Google Scholar 

  15. Zhao, X., Muller, E. G. & Rothstein, R. A suppressor of two essential checkpoint genes identifies a novel protein that negatively affects dNTP pools. Mol. Cell 2, 329–340 (1998).

    Article  CAS  Google Scholar 

  16. Newlon, C. S. et al. Analysis of replication origin function on chromosome III of Saccharomyces cerevisiae. Cold Spring Harb. Symp. Quant. Biol. 58, 415–423 (1993).

    Article  CAS  Google Scholar 

  17. Friedman, K. L. & Brewer, B. J. Analysis of replication intermediates by two-dimensional agarose gel electrophoresis. Methods Enzymol. 262, 613–627 (1995).

    Article  CAS  Google Scholar 

  18. Lindsay, H. D. et al. S-phase-specific activation of Cds1 kinase defines a subpathway of the checkpoint response in Schizosaccharomyces pombe. Genes Dev. 12, 382–395 (1998).

    Article  CAS  Google Scholar 

  19. Edwards, R. J., Bentley, N. J. & Carr, A. M. A Rad3–Rad26 complex response to DNA damage independently of other checkpoint proteins. Nature Cell Biol. 1, 393–398 (1999).

    Article  CAS  Google Scholar 

  20. Dimitrova, D. S. & Gilbert, D. M. Temporally coordinated assembly and disassembly of replication factories in the absence of DNA synthesis. Nature Cell Biol. 2, 686–694 (2000).

    Article  CAS  Google Scholar 

  21. Seigneur, M., Bidnenko, V., Ehrlich, S. D. & Michel, B. RuvAB acts at arrested replication forks. Cell 95, 419–430 (1998).

    Article  CAS  Google Scholar 

  22. Rothstein, R., Michel, B. & Gangloff, S. Replication fork pausing and recombination or “gimme a break”. Genes Dev. 14, 1–10 (2000).

    Article  CAS  Google Scholar 

  23. Martin-Parras, L., Hernandez, P., Martinez-Robles, M. L. & Schwartzman, J. B. Initiation of DNA replication in ColE1 plasmids containing multiple potential origins of replication. J. Biol. Chem. 267, 22496–22505 (1992).

    Article  CAS  Google Scholar 

  24. Kalejta, R. F. & Hamlin, J. L. Composite patterns in neutral/neutral two-dimensional gels demonstrate inefficient replication origin usage. Mol. Cell. Biol. 16, 4915–4922 (1996).

    Article  CAS  Google Scholar 

  25. Deshpande, A. M. & Newlon, C. S. DNA replication fork pause sites dependent on transcription. Science 272, 1030–1033 (1996).

    Article  ADS  CAS  Google Scholar 

  26. Ivessa, A. S., Zhou, J. Q. & Zakian, V. A. The Saccharomyces Pif1p DNA helicase and the highly related Rrm3p have opposite effects on replication fork progression in ribosomal DNA. Cell 100, 479–489 (2000).

    Article  CAS  Google Scholar 

  27. Thomas, B. J. & Rothstein, R. Elevated recombination rates in transcriptionally active DNA. Cell 56, 619–629 (1989).

    Article  CAS  Google Scholar 

  28. Marini, F. et al. Role for DNA primase in coupling DNA replication to DNA damage response. EMBO J. 16, 639–650 (1997).

    Article  CAS  Google Scholar 

  29. Pellicioli, A., Lee, S. E., Lucca, C., Foiani, M. & Haber, J. E. Regulation of Saccharomyces Rad53 checkpoint kinase during adaptation from DNA damage-induced G2/M arrest. Mol. Cell 7, 293–300 (2001).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank S. Alberti, L. Fabiani, A. Gambetta, S. Pintus and J. Theis for technical advice and support. We also thank A. Carr, J. Diffley, J. Haber, R. Rothstein, J. Sogo and all the members of our laboratory for helpful discussions. This work was supported by Associazione Italiana per la Ricerca sul Cancro and partially by grants from Telethon–Italy, Cofinanziamento MURST–Università di Milano, MURST (5%) Biomolecole per la Salute Umana, and CNR Target Project on Biotechnology, by a EU TMR contract, and by a NIH grant to C.S.N.

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Authors and Affiliations

  1. Istituto F.I.R.C. di Oncologia Molecolare, Via Serio 21, 20141, Milano, Italy

    Massimo Lopes, Cecilia Cotta-Ramusino, Achille Pellicioli, Giordano Liberi, Paolo Plevani, Marco Muzi-Falconi & Marco Foiani

  2. Dipartimento di Genetica e di Biologia dei Microrganismi, Università degli Studi di Milano, Via Celoria 26, 20133, Milano, Italy

    Massimo Lopes, Cecilia Cotta-Ramusino, Achille Pellicioli, Giordano Liberi, Paolo Plevani, Marco Muzi-Falconi & Marco Foiani

  3. Department of Microbiology & Molecular Genetics, UMDNJ—New Jersey Medical School, Newark, 07103, New Jersey, USA

    Carol S. Newlon

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Correspondence to Marco Foiani.

Supplementary information

Addentum to Figure 1

(JPG 27.8 KB)

Protein extracts prepared from cells taken at the indicated time points were analysed by western blotting using anti-Rad53 antibodies. The DNA content of wt and rad53 cells was analysed at the indicated time points by FACS analysis.

Addentum to Figure 4

(JPG 10.5 KB)

Protein extracts prepared from cells taken at the indicated time points were analysed by western blotting using anti-DNA polymerase alpha B subunit antibodies. The appearence of the hyperphosphorylated form of the B subunit indicates that the checkpoint has been inactivated (for further details see ref.6). The DNA content of wt and rad53 cells was analysed at the indicated time points by FACS analysis.

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Lopes, M., Cotta-Ramusino, C., Pellicioli, A. et al. The DNA replication checkpoint response stabilizes stalled replication forks. Nature 412, 557–561 (2001). https://doi.org/10.1038/35087613

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