Radiation and Environmental Biophysics

, Volume 46, Issue 4, pp 401–407 | Cite as

Roles of Saccharomyces cerevisiae RAD17 and CHK1 checkpoint genes in the repair of double-strand breaks in cycling cells

  • Nelson Bracesco
  • Ema C. Candreva
  • Deborah Keszenman
  • Ana G. Sánchez
  • Sandra Soria
  • Mercedes Dell
  • Wolfram Siede
  • Elia NunesEmail author
Original Paper


Checkpoints are components of signalling pathways involved in genome stability. We analysed the putative dual functions of Rad17 and Chk1 as checkpoints and in DNA repair using mutant strains of Saccharomyces cerevisiae. Logarithmic populations of the diploid checkpoint-deficient mutants, chk1Δ/chk1Δ and rad17Δ/rad17Δ, and an isogenic wild-type strain were exposed to the radiomimetic agent bleomycin (BLM). DNA double-strand breaks (DSBs) determined by pulsed-field electrophoresis, surviving fractions, and proliferation kinetics were measured immediately after treatments or after incubation in nutrient medium in the presence or absence of cycloheximide (CHX). The DSBs induced by BLM were reduced in the wild-type strain as a function of incubation time after treatment, with chromosomal repair inhibited by CHX. rad17Δ/rad17Δ cells exposed to low BLM concentrations showed no DSB repair, low survival, and CHX had no effect. Conversely, rad17Δ/rad17Δ cells exposed to high BLM concentrations showed DSB repair inhibited by CHX. chk1Δ/chk1Δ cells showed DSB repair, and CHX had no effect; these cells displayed the lowest survival following high BLM concentrations. Present results indicate that Rad17 is essential for inducible DSB repair after low BLM-concentrations (low levels of oxidative damage). The observations in the chk1Δ/chk1Δ mutant strain suggest that constitutive nonhomologous end-joining is involved in the repair of BLM-induced DSBs. The differential expression of DNA repair and survival in checkpoint mutants as compared to wild-type cells suggests the presence of a regulatory switch-network that controls and channels DSB repair to alternative pathways, depending on the magnitude of the DNA damage and genetic background.


Mutant Strain Proliferation Kinetic Liquid Nutrient Medium Solid Nutrient Medium Cell Proliferation Kinetic 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



This work was supported by NIH/Fogarty International Research Collaboration Award TW01189. We are indebted to Lourdes Blanc for excellent technical assistance, to PEDECIBA (Uruguay) for partial support, and to Dr. Anna Friedl for comments on the manuscript.


  1. 1.
    Hartwell L, Weinert TA (1989) Checkpoints: controls that ensure the order of cell cycle events. Science 246:629–634CrossRefADSGoogle Scholar
  2. 2.
    Elledge SJ (1996) Cell cycle checkpoints: preventing an identity crisis. Science 274:1664–1672CrossRefADSGoogle Scholar
  3. 3.
    Nyberg KA, Michelson RJ, Putnam CW, Weinert TA (2002) Toward maintaining the genome: DNA damage and replication checkpoint. Annu Rev Genet 36:617–656CrossRefGoogle Scholar
  4. 4.
    Rhind N, Russel P (2000) Chk1 and Cds1: linchpins of the DNA damage and replication checkpoint pathway. J Cell Sci 113:3889–3896Google Scholar
  5. 5.
    Bashkirov V, King J, Bashkirova E, Schmuckli-Maurer J, Heyer W (2000) DNA Repair protein Rad55 is a terminal substrate of the DNA damage checkpoints. Mol Cell Biol 20:4393–4404CrossRefGoogle Scholar
  6. 6.
    Mills K, Sinclair DA, Guarente L (1999) MEC1-dependent redistribution of the Sir3 silencing protein from telomeres to DNA double-strand breaks. Cell 97:609–620CrossRefGoogle Scholar
  7. 7.
    Lowndes NF, Murguia RJ (2000) Sensing and responding to DNA damage. Curr Opin Genet Dev 10:17–25CrossRefGoogle Scholar
  8. 8.
    Shin DS, Chahwan C, Huffman JL, Tainer JA (2004) Structure and function of the double-strand break repair machinery. DNA Repair 3:863–873CrossRefGoogle Scholar
  9. 9.
    Zhang H, Zhu Z, Vidanes G, Mbangkollo D, Liu Y, Siede W (2001) Characterization of DNA damage-stimulated self-interaction of Saccharomyces cerevisiae checkpoint protein Rad17p. J Biol Chem 276:16715–16723Google Scholar
  10. 10.
    Majka J, Burgers PM (2003) Yeast Rad17/Mec3/Ddc1: a sliding clamp for the DNA damage checkpoint. Proc Natl Acad Sci USA 100:2249–2254CrossRefADSGoogle Scholar
  11. 11.
    Syljuasen RG, Storgaard Sorensen C, Tengbjerg Hansen L, Fugger K, Lundin C, Johansson F, Helleday F, Sehested T, Lukas M, Bartek J (2005) Inhibition of human Chk1 causes increased initiation of DNA replication, phosphorylation of ATR targets, and DNA breakage. Mol Cell Biol 25:3553–3562CrossRefGoogle Scholar
  12. 12.
    Moore CW (1978) Bleomycin-induced mutation and recombination in Saccharomyces cerevisiae. Mutat Res 58:41–49CrossRefGoogle Scholar
  13. 13.
    Severgnini AA, Lillo OL, Nunes E (1991) Analysis of bleomycin-induced mutagenic function related to the PSO4(=XS9) gene of Saccharomyces cerevisiae. Environ Mol Mutagen 18:102–106CrossRefGoogle Scholar
  14. 14.
    Lillo OL, Severgnini AA, Nunes E (1997) Interactive lethal and mutagenic effects of ultraviolet light and bleomycin in yeast: synergism or antagonism? Radiat Res 148:476–480CrossRefGoogle Scholar
  15. 15.
    Moore CW, McKoy J, Dardalhon M, Davermann D, Martínez M, Averbeck D (2000) DNA damage-inducible and RAD52-independent repair on DNA double-strand breaks in Saccharomyces cerevisiae. Genetics 154:1085–1099Google Scholar
  16. 16.
    Keszenman DJ, Candreva EC, Nunes E (2000) Cellular and molecular effects of bleomycin are modulated by heat shock in Sacharomyces cerevisiae. Mutat Res 459:29–41Google Scholar
  17. 17.
    Keszenman DJ, Candreva EC, Sánchez AG, Nunes E (2005) RAD6 gene is involved in heat shock induction of bleomycin resistance in Saccharomyces cerevisiae. Environ Mol Mutagen 45:36–43CrossRefGoogle Scholar
  18. 18.
    Einhorn LH, Donohue J (2002) Cis-diamminedichloroplatinum, vinblastine, and bleomycin combination chemotherapy in disseminated testicular cancer. J Urol 167:928–932CrossRefGoogle Scholar
  19. 19.
    Friedl A, Kiechle M, Fellerhoff B, Eckardt-Schupp F (1998) Radiation-induced chromosome aberrations in S. cerevisiae: influence of DNA repair pathways. Genetics 148:975–988Google Scholar
  20. 20.
    Zhang H, Zhu Z, Vidanes G, Mbangkollo D, Liu Y, Siede W (2001) Characterization of DNA damage-stimulated self-interaction of Saccharomyces cerevisiae checkpoint protein Rad17p. J Biol Chem 276:16715–16723Google Scholar
  21. 21.
    Siede W, Eckardt F, Brendel M (1983) Analysis of mutagenic DNA repair in a thermoconditional repair mutant of Saccharomyces cerevisiae. II. Influence of cycloheximide on UV-irradiated exponentially growing rev2ts cells. Mol Gen Genet 190(3):413–416CrossRefGoogle Scholar
  22. 22.
    Geigl EM, Eckardt-Schupp F (1991) The repair of double-strand breaks and S1 nuclease-sensitive sites can be monitored chromosome-specifically in Saccharomyces cerevisiae using pulsed-field gel electrophoresis. Mol Microbiol 5:1615–1620CrossRefGoogle Scholar
  23. 23.
    Baur M (1990) Analyse der Rolle von Glutathion bei der Induktion und Reparatur von Doppelstrangbrueche mit Hilfe der Puls-Feld-Gelelektrophorese in Hefe. PhD Thesis, L-Maximilians University, MunichGoogle Scholar
  24. 24.
    Cole GM, Schild D, Lovett ST, Mortimer RK (1987) Regulation of RAD54- and RAD52-lacZ gene fusions in Saccharomyces cerevisiae in response to DNA damage. Mol Cell Biol 7:1078–1084Google Scholar
  25. 25.
    Ulrich H (2002) Degradation or maintenance: actions of the ubiquitin system on eukaryotic chromatin. Euk Cell 1:1–10CrossRefGoogle Scholar
  26. 26.
    Keszenman DJ, Salvo VA, Nunes E (1992) Effects of bleomycin on growth kinetics and survival of Saccharomyces cerevisiae: a model of repair pathways. J Bacteriol 174:3125-3132Google Scholar
  27. 27.
    Candreva E, Keszenman DJ, Bracesco N, Soria S, Sánchez AG, Dell M, Siede W, Nunes E (2004) A role of S. cerevisiae checkpoint protein Rad17 but not Chk1 in the repair of bleomycin-induced double-strand breaks in cycling and non-cycling cells. In: DNA repair and mutagenesis: from molecular structure to biological consequences. ASM Conferences, Southampton, p 228Google Scholar
  28. 28.
    Friedberg EC, Walker GC, Siede W, Wood RD, Schultz RA, Ellenberger T (2006) In: DNA repair and mutagenesis, 2nd edn. ASM Press, Washington, pp 724–735Google Scholar
  29. 29.
    Siede W, Friedl AA, Dianova I, Eckardt-Schupp F, Friedberg EC (1996) The Saccharomyces cerevisiae Ku autoantigen homologue affects radiosensitivity only in the abscence of homologous recombination. Genetics 142:91–102Google Scholar
  30. 30.
    Letavayová L, Marková E, Hermanská K, Vlcková V, Vlasáková D, Chovanec M, Brozmanová J (2006) Relative contribution of homologous recombination and non-homologous end-joining to DNA double-strand break repair after oxidative stress in Saccharomyces cerevisiae. DNA Repair 5:602–610CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Nelson Bracesco
    • 1
  • Ema C. Candreva
    • 1
  • Deborah Keszenman
    • 1
  • Ana G. Sánchez
    • 1
  • Sandra Soria
    • 1
  • Mercedes Dell
    • 1
  • Wolfram Siede
    • 2
  • Elia Nunes
    • 1
    Email author
  1. 1.Lab. Radiobiología, Departamento Biofísica, Facultad de MedicinaUniversidad de la RepúblicaMontevideoUruguay
  2. 2.Department of Cell Biology and GeneticsUniversity of North Texas Health Science CenterFort WorthUSA

Personalised recommendations