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. 1996 Aug 1;15(15):3912–3922.

A novel role for the budding yeast RAD9 checkpoint gene in DNA damage-dependent transcription.

A Aboussekhra 1, J E Vialard 1, D E Morrison 1, M A de la Torre-Ruiz 1, L Cernáková 1, F Fabre 1, N F Lowndes 1
PMCID: PMC452098  PMID: 8670896

Abstract

Cells respond to DNA damage by arresting cell cycle progression and activating several DNA repair mechanisms. These responses allow damaged DNA to be repaired efficiently, thus ensuring the maintenance of genetic integrity. In the budding yeast, Saccharomyces cerevisiae, DNA damage leads both to activation of checkpoints at the G1, S and G2 phases of the cell cycle and to a transcriptional response. The G1 and G2 checkpoints have been shown previously to be under the control of the RAD9 gene. We show here that RAD9 is also required for the transcriptional response to DNA damage. Northern blot analysis demonstrated that RAD9 controls the DNA damage-specific induction of a large 'regulon' of repair, replication and recombination genes. This induction is cell-cycle independent as it was observed in asynchronous cultures and cells blocked in G1 or G2/M. RAD9-dependent induction was also observed from isolated damage responsive promoter elements in a lacZ reporter-based plasmid assay. RAD9 cells deficient in the transcriptional response were more sensitive to DNA damage than wild-type cells, even after functional substitution of checkpoints, suggesting that this activation may have an important role in DNA repair. Our findings parallel observations with the Escherichia coli SOS system and suggest the existence of an analogous eukaryotic network coordinating the cellular responses to DNA damage.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aboussekhra A., Chanet R., Adjiri A., Fabre F. Semidominant suppressors of Srs2 helicase mutations of Saccharomyces cerevisiae map in the RAD51 gene, whose sequence predicts a protein with similarities to procaryotic RecA proteins. Mol Cell Biol. 1992 Jul;12(7):3224–3234. doi: 10.1128/mcb.12.7.3224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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. 1994 Oct 15;8(20):2401–2415. doi: 10.1101/gad.8.20.2401. [DOI] [PubMed] [Google Scholar]
  3. Basile G., Aker M., Mortimer R. K. Nucleotide sequence and transcriptional regulation of the yeast recombinational repair gene RAD51. Mol Cell Biol. 1992 Jul;12(7):3235–3246. doi: 10.1128/mcb.12.7.3235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cole G. M., Mortimer R. K. Failure to induce a DNA repair gene, RAD54, in Saccharomyces cerevisiae does not affect DNA repair or recombination phenotypes. Mol Cell Biol. 1989 Aug;9(8):3314–3322. doi: 10.1128/mcb.9.8.3314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Elledge S. J., Davis R. W. DNA damage induction of ribonucleotide reductase. Mol Cell Biol. 1989 Nov;9(11):4932–4940. doi: 10.1128/mcb.9.11.4932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Elledge S. J., Davis R. W. Identification of the DNA damage-responsive element of RNR2 and evidence that four distinct cellular factors bind it. Mol Cell Biol. 1989 Dec;9(12):5373–5386. doi: 10.1128/mcb.9.12.5373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Game J. C. DNA double-strand breaks and the RAD50-RAD57 genes in Saccharomyces. Semin Cancer Biol. 1993 Apr;4(2):73–83. [PubMed] [Google Scholar]
  8. Guarente L., Mason T. Heme regulates transcription of the CYC1 gene of S. cerevisiae via an upstream activation site. Cell. 1983 Apr;32(4):1279–1286. doi: 10.1016/0092-8674(83)90309-4. [DOI] [PubMed] [Google Scholar]
  9. Guzder S. N., Habraken Y., Sung P., Prakash L., Prakash S. Reconstitution of yeast nucleotide excision repair with purified Rad proteins, replication protein A, and transcription factor TFIIH. J Biol Chem. 1995 Jun 2;270(22):12973–12976. doi: 10.1074/jbc.270.22.12973. [DOI] [PubMed] [Google Scholar]
  10. Habraken Y., Sung P., Prakash L., Prakash S. Yeast excision repair gene RAD2 encodes a single-stranded DNA endonuclease. Nature. 1993 Nov 25;366(6453):365–368. doi: 10.1038/366365a0. [DOI] [PubMed] [Google Scholar]
  11. Hartwell L. H., Weinert T. A. Checkpoints: controls that ensure the order of cell cycle events. Science. 1989 Nov 3;246(4930):629–634. doi: 10.1126/science.2683079. [DOI] [PubMed] [Google Scholar]
  12. Hurd H. K., Roberts J. W. Upstream regulatory sequences of the yeast RNR2 gene include a repression sequence and an activation site that binds the RAP1 protein. Mol Cell Biol. 1989 Dec;9(12):5359–5372. doi: 10.1128/mcb.9.12.5359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Johnston L. H., Johnson A. L. The DNA repair genes RAD54 and UNG1 are cell cycle regulated in budding yeast but MCB promoter elements have no essential role in the DNA damage response. Nucleic Acids Res. 1995 Jun 25;23(12):2147–2152. doi: 10.1093/nar/23.12.2147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Johnston L. H., Lowndes N. F. Cell cycle control of DNA synthesis in budding yeast. Nucleic Acids Res. 1992 May 25;20(10):2403–2410. doi: 10.1093/nar/20.10.2403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kastan M. B., Zhan Q., el-Deiry W. S., Carrier F., Jacks T., Walsh W. V., Plunkett B. S., Vogelstein B., Fornace A. J., Jr A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell. 1992 Nov 13;71(4):587–597. doi: 10.1016/0092-8674(92)90593-2. [DOI] [PubMed] [Google Scholar]
  16. Kuerbitz S. J., Plunkett B. S., Walsh W. V., Kastan M. B. Wild-type p53 is a cell cycle checkpoint determinant following irradiation. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7491–7495. doi: 10.1073/pnas.89.16.7491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lindahl T. Instability and decay of the primary structure of DNA. Nature. 1993 Apr 22;362(6422):709–715. doi: 10.1038/362709a0. [DOI] [PubMed] [Google Scholar]
  18. Lowndes N. F., Johnson A. L., Johnston L. H. Coordination of expression of DNA synthesis genes in budding yeast by a cell-cycle regulated trans factor. Nature. 1991 Mar 21;350(6315):247–250. doi: 10.1038/350247a0. [DOI] [PubMed] [Google Scholar]
  19. Morrison D. P., Hastings P. J. Characterization of the mutator mutation mut5-1. Mol Gen Genet. 1979 Aug;175(1):57–65. doi: 10.1007/BF00267856. [DOI] [PubMed] [Google Scholar]
  20. Naumovski L., Friedberg E. C. Saccharomyces cerevisiae RAD2 gene: isolation, subcloning, and partial characterization. Mol Cell Biol. 1984 Feb;4(2):290–295. doi: 10.1128/mcb.4.2.290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Painter R. B., Young B. R. Radiosensitivity in ataxia-telangiectasia: a new explanation. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7315–7317. doi: 10.1073/pnas.77.12.7315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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. 1995 Sep 8;82(5):841–847. doi: 10.1016/0092-8674(95)90481-6. [DOI] [PubMed] [Google Scholar]
  23. Rothstein R. J. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. doi: 10.1016/0076-6879(83)01015-0. [DOI] [PubMed] [Google Scholar]
  24. Saeki T., Machida I., Nakai S. Genetic control of diploid recovery after gamma-irradiation in the yeast Saccharomyces cerevisiae. Mutat Res. 1980 Dec;73(2):251–265. doi: 10.1016/0027-5107(80)90192-x. [DOI] [PubMed] [Google Scholar]
  25. Schiestl R. H., Reynolds P., Prakash S., Prakash L. Cloning and sequence analysis of the Saccharomyces cerevisiae RAD9 gene and further evidence that its product is required for cell cycle arrest induced by DNA damage. Mol Cell Biol. 1989 May;9(5):1882–1896. doi: 10.1128/mcb.9.5.1882. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Shinohara A., Ogawa H., Ogawa T. Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein. Cell. 1992 May 1;69(3):457–470. doi: 10.1016/0092-8674(92)90447-k. [DOI] [PubMed] [Google Scholar]
  27. Siede W., Friedberg A. S., Dianova I., Friedberg E. C. Characterization of G1 checkpoint control in the yeast Saccharomyces cerevisiae following exposure to DNA-damaging agents. Genetics. 1994 Oct;138(2):271–281. doi: 10.1093/genetics/138.2.271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Siede W., Friedberg A. S., Friedberg E. C. RAD9-dependent G1 arrest defines a second checkpoint for damaged DNA in the cell cycle of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1993 Sep 1;90(17):7985–7989. doi: 10.1073/pnas.90.17.7985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Siede W., Friedberg E. C. Regulation of the yeast RAD2 gene: DNA damage-dependent induction correlates with protein binding to regulatory sequences and their deletion influences survival. Mol Gen Genet. 1992 Mar;232(2):247–256. doi: 10.1007/BF00280003. [DOI] [PubMed] [Google Scholar]
  30. Siede W., Robinson G. W., Kalainov D., Malley T., Friedberg E. C. Regulation of the RAD2 gene of Saccharomyces cerevisiae. Mol Microbiol. 1989 Dec;3(12):1697–1707. doi: 10.1111/j.1365-2958.1989.tb00155.x. [DOI] [PubMed] [Google Scholar]
  31. Simon J. A., Lis J. T. A germline transformation analysis reveals flexibility in the organization of heat shock consensus elements. Nucleic Acids Res. 1987 Apr 10;15(7):2971–2988. doi: 10.1093/nar/15.7.2971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Singh K. K., Samson L. Replication protein A binds to regulatory elements in yeast DNA repair and DNA metabolism genes. Proc Natl Acad Sci U S A. 1995 May 23;92(11):4907–4911. doi: 10.1073/pnas.92.11.4907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sung P. Catalysis of ATP-dependent homologous DNA pairing and strand exchange by yeast RAD51 protein. Science. 1994 Aug 26;265(5176):1241–1243. doi: 10.1126/science.8066464. [DOI] [PubMed] [Google Scholar]
  34. Sung P., Robberson D. L. DNA strand exchange mediated by a RAD51-ssDNA nucleoprotein filament with polarity opposite to that of RecA. Cell. 1995 Aug 11;82(3):453–461. doi: 10.1016/0092-8674(95)90434-4. [DOI] [PubMed] [Google Scholar]
  35. Walker G. C. Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli. Microbiol Rev. 1984 Mar;48(1):60–93. doi: 10.1128/mr.48.1.60-93.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wang Z., Wu X., Friedberg E. C. Nucleotide-excision repair of DNA in cell-free extracts of the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):4907–4911. doi: 10.1073/pnas.90.11.4907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Weinert T. A., Hartwell L. H. Cell cycle arrest of cdc mutants and specificity of the RAD9 checkpoint. Genetics. 1993 May;134(1):63–80. doi: 10.1093/genetics/134.1.63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Weinert T. A., Hartwell L. H. Characterization of RAD9 of Saccharomyces cerevisiae and evidence that its function acts posttranslationally in cell cycle arrest after DNA damage. Mol Cell Biol. 1990 Dec;10(12):6554–6564. doi: 10.1128/mcb.10.12.6554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Weinert T. A., Hartwell L. H. The RAD9 gene controls the cell cycle response to DNA damage in Saccharomyces cerevisiae. Science. 1988 Jul 15;241(4863):317–322. doi: 10.1126/science.3291120. [DOI] [PubMed] [Google Scholar]
  40. Weinert T. A., Kiser G. L., Hartwell L. H. Mitotic checkpoint genes in budding yeast and the dependence of mitosis on DNA replication and repair. Genes Dev. 1994 Mar 15;8(6):652–665. doi: 10.1101/gad.8.6.652. [DOI] [PubMed] [Google Scholar]
  41. Werner-Washburne M., Braun E., Johnston G. C., Singer R. A. Stationary phase in the yeast Saccharomyces cerevisiae. Microbiol Rev. 1993 Jun;57(2):383–401. doi: 10.1128/mr.57.2.383-401.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. White J. H., Barker D. G., Nurse P., Johnston L. H. Periodic transcription as a means of regulating gene expression during the cell cycle: contrasting modes of expression of DNA ligase genes in budding and fission yeast. EMBO J. 1986 Jul;5(7):1705–1709. doi: 10.1002/j.1460-2075.1986.tb04414.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Zampetti-Bosseler F., Scott D. Cell death, chromosome damage and mitotic delay in normal human, ataxia telangiectasia and retinoblastoma fibroblasts after x-irradiation. Int J Radiat Biol Relat Stud Phys Chem Med. 1981 May;39(5):547–558. doi: 10.1080/09553008114550651. [DOI] [PubMed] [Google Scholar]
  44. Zhou Z., Elledge S. J. DUN1 encodes a protein kinase that controls the DNA damage response in yeast. Cell. 1993 Dec 17;75(6):1119–1127. doi: 10.1016/0092-8674(93)90321-g. [DOI] [PubMed] [Google Scholar]
  45. el-Deiry W. S., Tokino T., Velculescu V. E., Levy D. B., Parsons R., Trent J. M., Lin D., Mercer W. E., Kinzler K. W., Vogelstein B. WAF1, a potential mediator of p53 tumor suppression. Cell. 1993 Nov 19;75(4):817–825. doi: 10.1016/0092-8674(93)90500-p. [DOI] [PubMed] [Google Scholar]

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