Methods for Analyzing Checkpoint Responses in Caenorhabditis elegans

  • Anton Gartner
  • Amy J. MacQueen
  • Anne M. Villeneuve
Part of the Methods in Molecular Biology™ book series (MIMB, volume 280)

Abstract

In response to genotoxic insults, cells activate DNA damage checkpoint pathways that stimulate DNA repair, lead to a transient cell cycle arrest, and/or elicit programmed cell death (apoptosis) of affected cells. The Caenorhabditis elegans germ line was recently established as a model system to study these processes in a genetically tractable, multicellular organism. The utility of this system was revealed by the finding that upon treatment with genotoxic agents, premeiotic C. elegans germ cells transiently halt cell cycle progression, whereas meiotic prophase germ cells in the late pachytene stage readily undergo apoptosis. Further, accumulation of unrepaired meiotic recombination intermediates can also lead to the apoptotic demise of affected pachytene cells. DNA damage-induced cell death requires key components of the evolutionarily conserved apoptosis machinery. Moreover, both cell cycle arrest and pachytene apoptosis responses depend on conserved DNA damage checkpoint proteins. Genetics- and genomics-based approaches that have demonstrated roles for conserved checkpoint proteins have also begun to uncover novel components of these response pathways. In this chapter, we will briefly review the C. elegans DNA damage response field, and we will discuss in detail the methods that are being used to assay DNA damage responses in C. elegans.

Key Words

C. elegans C. elegans germ line C. elegans methods apoptosis programmed cell death DNA damage responses ced genes germ line checkpoint responses p53 cep-1 cell cycle arrest RNAi RNAi feeding co-suppression meiosis recombination 

References

  1. 1.
    Gartner, A., Milstein, S., Ahmed, S., Hodgkin, J., and Hengartner, M. O. (2000) A conserved checkpoint pathway mediates DNA damage-induced apoptosis and cell cycle arrest in C. elegans. Mol. Cell 5, 435–443.PubMedCrossRefGoogle Scholar
  2. 2.
    Seydoux, G. and Schedl, T. (2001) The germline in C. elegans: origins, proliferation, and silencing. Int. Rev. Cytol. 203, 139–185.PubMedCrossRefGoogle Scholar
  3. 3.
    Gumienny, T. L., Lambie, E., Hartwieg, E., Horvitz, H. R., and Hengartner, M. O. (1999) Genetic control of programmed cell death in the Caenorhabditis elegans hermaphrodite germline. Development 126, 1011–1022.PubMedGoogle Scholar
  4. 4.
    Keeney, S. (2001) Mechanism and control of meiotic recombination initiation. Curr. Top. Dev. Bol. 52, 1–53.CrossRefGoogle Scholar
  5. 5.
    Rinaldo, C., Bazzicalupo, P., Ederle, S., Hilliard, M., and La Volpe, A. (2002) Roles for Caenorhabditis elegans rad-51 in meiosis and in resistance to ionizing radiation during development. Genetics 160, 471–479.PubMedGoogle Scholar
  6. 6.
    Neufeld, T. P. and Edgar, B. A. (1998) Connections between growth and the cell cycle. Curr. Opin. Cell Biol. 10, 784–790.PubMedCrossRefGoogle Scholar
  7. 7.
    Weinert, T. A. and Hartwell, L. H. (1988) The RAD9 gene controls the cell cycle response to DNA damage in Saccharomyces cerevisiae. Science 241, 317–322.PubMedCrossRefGoogle Scholar
  8. 8.
    Dasika, G. K., Lin, S. C., Zhao, S., Sung, P., Tomkinson, A., and Lee, E. Y. (1999) DNA damage-induced cell cycle checkpoints and DNA strand break repair in development and tumorigenesis. Oncogene 18, 7883–7899.PubMedCrossRefGoogle Scholar
  9. 9.
    Murakami, H. and Nurse, P. (2000) DNA replication and damage checkpoints and meiotic cell cycle controls in the fission and budding yeasts. Biochem. J. 349, 1–12.PubMedCrossRefGoogle Scholar
  10. 10.
    Rhind, N. and Russell, P. (2000) Checkpoints: it takes more than time to heal some wounds. Curr. Biol. 10, R908–911.PubMedCrossRefGoogle Scholar
  11. 11.
    MacQueen, A. J. and Villeneuve, A. M. (2001) Nuclear reorganization and homologous chromosome pairing during meiotic prophase require C. elegans chk-2. Genes Dev. 15, 1674–1687.PubMedCrossRefGoogle Scholar
  12. 12.
    Ahmed, S., Alpi, A., Hengartner, M. O., and Gartner, A. (2001) C. elegans RAD-5/CLK-2 defines a new DNA damage checkpoint protein. Curr. Biol. 11, 1934–1944.PubMedCrossRefGoogle Scholar
  13. 13.
    Boulton, S. J., Gartner, A., Reboul, J., et al. (2002) Combined functional genomic maps of the C. elegans DNA damage response. Science 295, 127–131.PubMedCrossRefGoogle Scholar
  14. 14.
    Ahmed, S. and Hodgkin, J. (2000) MRT-2 checkpoint protein is required for germline immortality and telomere replication in C. elegans. Nature 403, 159–164.PubMedCrossRefGoogle Scholar
  15. 15.
    Kota, R. S. and Runge, K. W. (1998) The yeast telomere length regulator TEL2 encodes a protein that binds to telomeric DNA. Nucleic Acids Res. 26, 1528–1535.PubMedCrossRefGoogle Scholar
  16. 16.
    Schumacher, B., Hofmann, K., Boulton, S., and Gartner, A. (2001) The C. elegans homolog of the p53 tumor suppressor is required for DNA damage-induced apoptosis. Curr. Biol. 11, 1722–1727.PubMedCrossRefGoogle Scholar
  17. 17.
    Derry, W. B., Putzke, A. P., and Rothman, J. H. (2001) Caenorhabditis elegans p53: role in apoptosis, meiosis, and stress resistance. Science 294, 591–595.PubMedCrossRefGoogle Scholar
  18. 18.
    Hofmann, E. R., Milstein, S., Boulton, S. J., et al. (2002) Caenorhabditis elegans HUS-1 is a DNA damage checkpoint protein required for genome stability and EGL-1-mediated apoptosis. Curr. Biol. 12, 1908–1918.PubMedCrossRefGoogle Scholar
  19. 19.
    Sulston, J. and Hodgkin, J. (1988) Methods. In: The Nematode Caenorhabditis elegans (Wood, W. B., ed), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY: pp. 587–606.Google Scholar
  20. 20.
    Brenner, S. (1974) The genetics of Caenorhabditis elegans. Genetics 77, 71–94.PubMedGoogle Scholar
  21. 21.
    Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., and Mello, C. C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811.PubMedCrossRefGoogle Scholar
  22. 22.
    Ketting, R. F. and Plasterk, R. H. (2000) A genetic link between co-suppression and RNA interference in C. elegans. Nature 404, 296–298.PubMedCrossRefGoogle Scholar
  23. 23.
    Dernburg, A. F., Zalevsky, J., Colaiacovo, M. P., and Villeneuve, A. M. (2000) Transgene-mediated cosuppression in the C. elegans germ line. Genes Dev. 14, 1578–1583.PubMedGoogle Scholar
  24. 24.
    Tabara, H., Grishok, A., and Mello, C. C. (1998) RNAi in C. elegans: soaking in the genome sequence. Science 282, 430–431.PubMedCrossRefGoogle Scholar
  25. 25.
    Kamath, R. S., Martinez-Campos, M., Zipperlen, P., Fraser, A. G., and Ahringer, J. (2001) Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans. Genome Biol. 2, research 0002.1–0002.10.Google Scholar

Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  • Anton Gartner
    • 1
  • Amy J. MacQueen
    • 2
  • Anne M. Villeneuve
    • 3
  1. 1.Department of Cell BiologyMax Planck Institute for BiochemistryMartinsriedGermany
  2. 2.Department of Molecular BiologyUT Southwestern Medical CenterDallas
  3. 3.Departments of Developmental Biology and GeneticsStanford University School of MedicineStanford

Personalised recommendations