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

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Abstract

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.

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Acknowledgments

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.

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Authors and Affiliations

  1. Lab. Radiobiología, Departamento Biofísica, Facultad de Medicina, Universidad de la República, 11800, Montevideo, Uruguay

    Nelson Bracesco, Ema C. Candreva, Deborah Keszenman, Ana G. Sánchez, Sandra Soria, Mercedes Dell & Elia Nunes

  2. Department of Cell Biology and Genetics, University of North Texas Health Science Center, Fort Worth, TX, USA

    Wolfram Siede

Authors
  1. Nelson Bracesco
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  2. Ema C. Candreva
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  3. Deborah Keszenman
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  4. Ana G. Sánchez
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  5. Sandra Soria
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  6. Mercedes Dell
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  7. Wolfram Siede
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  8. Elia Nunes
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Correspondence to Elia Nunes.

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Nelson Bracesco and Ema C. Candreva have contributed equally to this article.

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Bracesco, N., Candreva, E.C., Keszenman, D. et al. Roles of Saccharomyces cerevisiae RAD17 and CHK1 checkpoint genes in the repair of double-strand breaks in cycling cells. Radiat Environ Biophys 46, 401–407 (2007). https://doi.org/10.1007/s00411-007-0119-y

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  • DOI: https://doi.org/10.1007/s00411-007-0119-y

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