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:

The Ino80 chromatin-remodeling enzyme regulates replisome function and stability

Nature Structural & Molecular Biology volume 15, pages 338–345 (2008)Cite this article

Abstract

Previous studies have demonstrated essential roles for ATP-dependent chromatin-remodeling and chromatin-modifying enzymes in gene transcription and DNA repair, but few studies have addressed how the replication machinery deals with chromatin. Here we show that the Ino80 remodeling enzyme is recruited to replication origins as cells enter S phase. Inducible degradation of Ino80 shows that it is required continuously for efficient progression of forks, especially when cells are confronted with low levels of replication stress. Furthermore, we show that stalling of replication forks in an ino80 mutant is a lethal event, and that much of the replication machinery dissociates from the stalled fork. Our data indicate that the chromatin-remodeling activity of Ino80 regulates efficient progression of replication forks and that Ino80 has a crucial role in stabilizing a stalled replisome to ensure proper restart of DNA replication.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: An ino80Δ mutant is hypersensitive to replication stress caused by dNTP depletion.
Figure 2: Ino80 is essential for progression of S phase during replication stress.
Figure 3: Rapid stalling of replication forks in the absence of Ino80.
Figure 4: Ino80 has a continuous role in DNA replication.
Figure 5: The Ino80 complex associates with replication forks.
Figure 6: The Ino80 complex is required for maintaining replisome integrity during replication stress.

Similar content being viewed by others

References

  1. Bystricky, K., Heun, P., Gehlen, L., Langowski, J. & Gasser, S.M. Long-range compaction and flexibility of interphase chromatin in budding yeast analyzed by high-resolution imaging techniques. Proc. Natl. Acad. Sci. USA 101, 16495–16500 (2004).

    Article  CAS  Google Scholar 

  2. Groth, A. et al. Regulation of replication fork progression through histone supply and demand. Science 318, 1928–1931 (2007).

    Article  CAS  Google Scholar 

  3. Osborn, A.J., Elledge, S.J. & Zou, L. Checking on the fork: the DNA-replication stress-response pathway. Trends Cell Biol. 12, 509–516 (2002).

    Article  CAS  Google Scholar 

  4. Branzei, D. & Foiani, M. Interplay of replication checkpoints and repair proteins at stalled replication forks. DNA Repair (Amst.) 6, 994–1003 (2007).

    Article  CAS  Google Scholar 

  5. Lopes, M. et al. The DNA replication checkpoint response stabilizes stalled replication forks. Nature 412, 557–561 (2001).

    Article  CAS  Google Scholar 

  6. Kolodner, R.D., Putnam, C.D. & Myung, K. Maintenance of genome stability in Saccharomyces cerevisiae. Science 297, 552–557 (2002).

    Article  CAS  Google Scholar 

  7. Franco, A.A., Lam, W.M., Burgers, P.M. & Kaufman, P.D. Histone deposition protein Asf1 maintains DNA replisome integrity and interacts with replication factor C. Genes Dev. 19, 1365–1375 (2005).

    Article  CAS  Google Scholar 

  8. Han, J., Zhou, H., Li, Z., Xu, R.M. & Zhang, Z. Acetylation of lysine 56 of histone H3 catalyzed by RTT109 and regulated by ASF1 is required for replisome integrity. J. Biol. Chem. 282, 28587–28596 (2007).

    Article  CAS  Google Scholar 

  9. Han, J. et al. Rtt109 acetylates histone H3 lysine 56 and functions in DNA replication. Science 315, 653–655 (2007).

    Article  CAS  Google Scholar 

  10. Driscoll, R., Hudson, A. & Jackson, S.P. Yeast Rtt109 promotes genome stability by acetylating histone H3 on lysine 56. Science 315, 649–652 (2007).

    Article  CAS  Google Scholar 

  11. Collins, S.R. et al. Functional dissection of protein complexes involved in yeast chromosome biology using a genetic interaction map. Nature 446, 806–810 (2007).

    Article  CAS  Google Scholar 

  12. Bao, Y. & Shen, X. Ino80 subfamily of chromatin remodeling complexes. Mutat. Res. 618, 18–29 (2007).

    Article  CAS  Google Scholar 

  13. Jonsson, Z.O., Jha, S., Wohlschlegel, J.A. & Dutta, A. Rvb1p/Rvb2p recruit Arp5p and assemble a functional Ino80 chromatin remodeling complex. Mol. Cell 16, 465–477 (2004).

    Article  CAS  Google Scholar 

  14. Morrison, A.J. et al. INO80 and γ-H2AX interaction links ATP-dependent chromatin remodeling to DNA damage repair. Cell 119, 767–775 (2004).

    Article  CAS  Google Scholar 

  15. van Attikum, H., Fritsch, O., Hohn, B. & Gasser, S.M. Recruitment of the Ino80 complex by H2A phosphorylation links ATP-dependent chromatin remodeling with DNA double-strand break repair. Cell 119, 777–788 (2004).

    Article  CAS  Google Scholar 

  16. Papamichos-Chronakis, M., Krebs, J.E. & Peterson, C.L. Interplay between Ino80 and Swr1 chromatin remodeling enzymes regulates cell cycle checkpoint adaptation in response to DNA damage. Genes Dev. 20, 2437–2449 (2006).

    Article  CAS  Google Scholar 

  17. Shen, X., Mizuguchi, G., Hamiche, A. & Wu, C. A chromatin remodelling complex involved in transcription and DNA processing. Nature 406, 541–544 (2000).

    Article  CAS  Google Scholar 

  18. D'Amours, D. & Jackson, S.P. The yeast Xrs2 complex functions in S phase checkpoint regulation. Genes Dev. 15, 2238–2249 (2001).

    Article  CAS  Google Scholar 

  19. Allen, J.B., Zhou, Z., Siede, W., Friedberg, E.C. & Elledge, S.J. The SAD1/RAD53 protein kinase controls multiple checkpoints and DNA damage-induced transcription in yeast. Genes Dev. 8, 2401–2415 (1994).

    Article  CAS  Google Scholar 

  20. Tercero, J.A., Longhese, M.P. & Diffley, J.F. A central role for DNA replication forks in checkpoint activation and response. Mol. Cell 11, 1323–1336 (2003).

    Article  CAS  Google Scholar 

  21. 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 

  22. Masai, H. & Arai, K. Cdc7 kinase complex: a key regulator in the initiation of DNA replication. J. Cell. Physiol. 190, 287–296 (2002).

    Article  CAS  Google Scholar 

  23. Jong, A.Y., Wang, B. & Zhang, S.Q. Pulsed field gel electrophoresis labeling method to study the pattern of Saccharomyces cerevisiae chromosomal DNA synthesis during the G1/S phase of the cell cycle. Anal. Biochem. 227, 32–39 (1995).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  25. Bell, S.P. & Dutta, A. DNA replication in eukaryotic cells. Annu. Rev. Biochem. 71, 333–374 (2002).

    Article  CAS  Google Scholar 

  26. Diffley, J.F. Regulation of early events in chromosome replication. Curr. Biol. 14, R778–R786 (2004).

    Article  CAS  Google Scholar 

  27. Tanaka, T. & Nasmyth, K. Association of RPA with chromosomal replication origins requires an Mcm protein, and is regulated by Rad53, and cyclin- and Dbf4-dependent kinases. EMBO J. 17, 5182–5191 (1998).

    Article  CAS  Google Scholar 

  28. Aparicio, O.M., Weinstein, D.M. & Bell, S.P. Components and dynamics of DNA replication complexes in S. cerevisiae: redistribution of MCM proteins and Cdc45p during S phase. Cell 91, 59–69 (1997).

    Article  CAS  Google Scholar 

  29. Cobb, J.A., Bjergbaek, L., Shimada, K., Frei, C. & Gasser, S.M. DNA polymerase stabilization at stalled replication forks requires Mec1 and the RecQ helicase Sgs1. EMBO J. 22, 4325–4336 (2003).

    Article  CAS  Google Scholar 

  30. Zou, L. & Elledge, S.J. Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science 300, 1542–1548 (2003).

    Article  CAS  Google Scholar 

  31. Johnson, A. & O'Donnell, M. Cellular DNA replicases: components and dynamics at the replication fork. Annu. Rev. Biochem. 74, 283–315 (2005).

    Article  CAS  Google Scholar 

  32. Moldovan, G.L., Pfander, B. & Jentsch, S. PCNA, the maestro of the replication fork. Cell 129, 665–679 (2007).

    Article  CAS  Google Scholar 

  33. Tsurimoto, T. & Stillman, B. Functions of replication factor C and proliferating-cell nuclear antigen: functional similarity of DNA polymerase accessory proteins from human cells and bacteriophage T4. Proc. Natl. Acad. Sci. USA 87, 1023–1027 (1990).

    Article  CAS  Google Scholar 

  34. Cobb, J.A. et al. Replisome instability, fork collapse, and gross chromosomal rearrangements arise synergistically from Mec1 kinase and RecQ helicase mutations. Genes Dev. 19, 3055–3069 (2005).

    Article  CAS  Google Scholar 

  35. Shroff, R. et al. Distribution and dynamics of chromatin modification induced by a defined DNA double-strand break. Curr. Biol. 14, 1703–1711 (2004).

    Article  CAS  Google Scholar 

  36. Ogiwara, H., Enomoto, T. & Seki, M. The INO80 chromatin remodeling complex functions in sister chromatid cohesion. Cell Cycle 6, 1090–1095 (2007).

    Article  CAS  Google Scholar 

  37. Collins, N. et al. An ACF1-ISWI chromatin-remodeling complex is required for DNA replication through heterochromatin. Nat. Genet. 32, 627–632 (2002).

    Article  CAS  Google Scholar 

  38. Fan, J.Y., Gordon, F., Luger, K., Hansen, J.C. & Tremethick, D.J. The essential histone variant H2A.Z regulates the equilibrium between different chromatin conformational states. Nat. Struct. Biol. 9, 172–176 (2002).

    Article  CAS  Google Scholar 

  39. Sandell, L.L. & Zakian, V.A. Loss of a yeast telomere: arrest, recovery, and chromosome loss. Cell 75, 729–739 (1993).

    Article  CAS  Google Scholar 

  40. Longtine, M.S. et al. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953–961 (1998).

    Article  CAS  Google Scholar 

  41. Papamichos-Chronakis, M., Petrakis, T., Ktistaki, E., Topalidou, I. & Tzamarias, D. Cti6, a PHD domain protein, bridges the Cyc8-Tup1 corepressor and the SAGA coactivator to overcome repression at GAL1. Mol. Cell 9, 1297–1305 (2002).

    Article  CAS  Google Scholar 

  42. Liberi, G. et al. Methods to study replication fork collapse in budding yeast. Methods Enzymol. 409, 442–462 (2006).

    Article  CAS  Google Scholar 

  43. Oshiro, G., Owens, J.C., Shellman, Y., Sclafani, R.A. & Li, J.J. Cell cycle control of Cdc7p kinase activity through regulation of Dbf4p stability. Mol. Cell. Biol. 19, 4888–4896 (1999).

    Article  CAS  Google Scholar 

  44. Nougarede, R., Della Seta, F., Zarzov, P. & Schwob, E. Hierarchy of S-phase-promoting factors: yeast Dbf4-Cdc7 kinase requires prior S-phase cyclin-dependent kinase activation. Mol. Cell. Biol. 20, 3795–3806 (2000).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We are grateful to P. Kaufman (University of Massachusetts Medical School (UMMS), Worcester, Massachusetts) for antibodies to PCNA and for comments on the manuscript, to V. Zakian (Princeton University, Princeton, New Jersey) for strain LS20, and to A. Dutta (University of Virginia, Charlotte, Virginia) for the ino80-td strain. We also thank M. Marinus (UMMS) and N. Willis (UMMS) for assistance with the PFGE and two-dimensinal gel analysis, respectively. This work was supported by the US National Institutes of Health.

Author information

Authors and Affiliations

  1. Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation Street, Biotechnology 2, Suite 210, Worcester, 01605, Massachusetts, USA

    Manolis Papamichos-Chronakis & Craig L Peterson

Authors
  1. Manolis Papamichos-Chronakis
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Craig L Peterson
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

All experiments were designed and executed by M.P.-C., and C.L.P. helped with data interpretation; C.L.P. and M.P.-C. wrote the manuscript together.

Corresponding author

Correspondence to Craig L Peterson.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 (PDF 574 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Papamichos-Chronakis, M., Peterson, C. The Ino80 chromatin-remodeling enzyme regulates replisome function and stability. Nat Struct Mol Biol 15, 338–345 (2008). https://doi.org/10.1038/nsmb.1413

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.1413

This article is cited by

Search

Advanced 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