Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1996 May 1;24(9):1669–1675. doi: 10.1093/nar/24.9.1669

Cloning and characterization of RAD17, a gene controlling cell cycle responses to DNA damage in Saccharomyces cerevisiae.

W Siede 1, G Nusspaumer 1, V Portillo 1, R Rodriguez 1, E C Friedberg 1
PMCID: PMC145842  PMID: 8649984

Abstract

Mutants of the yeast Saccharomyces cerevisiae defective in the RAD17 gene are sensitive to ultraviolet (UV) and gamma radiation and manifest a defect in G2 arrest following radiation treatment. We have cloned the RAD17 gene by complementation of the UV sensitivity of a rad17-1 mutant and identified an ORF of 1.2 kb encoding a predicted gene product of 45.4 kDa with homology to the Schizosaccharomyces pombe rad1+ gene product and to Ustilago maydis Rec1, a known 3'->5'exonuclease. The RAD17 transcript is cell cycle regulated, with maximum steady-state levels during late G1. The rad17-1 mutation represents a missense mutation that maps to a conserved region of the gene. A rad17 disruption mutant grows normally and manifests levels of UV sensitivity similar that of the rad17-1 strain. As previously observed with other genes involved in G2 arrest (such as RAD9 and RAD24), RAD17 regulates radiation-induced G1 checkpoints at at least two possible arrest stages. One is equivalent to or upstream of START, the other at or downstream of the Cdc4 execution point. However, the temperature sensitivity of the cell cycle mutant dna1-1 (a G1 arrest mutant) is not influenced by inactivation of RAD17.

Full Text

The Full Text of this article is available as a PDF (115.0 KB).

Selected References

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

  1. 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]
  2. Aves S. J., Durkacz B. W., Carr A., Nurse P. Cloning, sequencing and transcriptional control of the Schizosaccharomyces pombe cdc10 'start' gene. EMBO J. 1985 Feb;4(2):457–463. doi: 10.1002/j.1460-2075.1985.tb03651.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barker D. G., White J. H., Johnston L. H. The nucleotide sequence of the DNA ligase gene (CDC9) from Saccharomyces cerevisiae: a gene which is cell-cycle regulated and induced in response to DNA damage. Nucleic Acids Res. 1985 Dec 9;13(23):8323–8337. doi: 10.1093/nar/13.23.8323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Carr A. M. Radiation checkpoints in model systems. Int J Radiat Biol. 1994 Dec;66(6 Suppl):S133–S139. [PubMed] [Google Scholar]
  6. Cox B. S., Parry J. M. The isolation, genetics and survival characteristics of ultraviolet light-sensitive mutants in yeast. Mutat Res. 1968 Jul-Aug;6(1):37–55. doi: 10.1016/0027-5107(68)90101-2. [DOI] [PubMed] [Google Scholar]
  7. Dirick L., Böhm T., Nasmyth K. Roles and regulation of Cln-Cdc28 kinases at the start of the cell cycle of Saccharomyces cerevisiae. EMBO J. 1995 Oct 2;14(19):4803–4813. doi: 10.1002/j.1460-2075.1995.tb00162.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Eberly S. L., Sakai A., Sugino A. Mapping and characterizing a new DNA replication mutant in Saccharomyces cerevisiae. Yeast. 1989 Mar-Apr;5(2):117–129. doi: 10.1002/yea.320050207. [DOI] [PubMed] [Google Scholar]
  9. Elledge S. J., Davis R. W. Two genes differentially regulated in the cell cycle and by DNA-damaging agents encode alternative regulatory subunits of ribonucleotide reductase. Genes Dev. 1990 May;4(5):740–751. doi: 10.1101/gad.4.5.740. [DOI] [PubMed] [Google Scholar]
  10. Enoch T., Carr A. M., Nurse P. Fission yeast genes involved in coupling mitosis to completion of DNA replication. Genes Dev. 1992 Nov;6(11):2035–2046. doi: 10.1101/gad.6.11.2035. [DOI] [PubMed] [Google Scholar]
  11. Evans D. R., Singer R. A., Johnston G. C., Wheals A. E. Cell-cycle mutations among the collection of Saccharomyces cerevisiae dna mutants. FEMS Microbiol Lett. 1994 Feb 15;116(2):147–153. doi: 10.1111/j.1574-6968.1994.tb06693.x. [DOI] [PubMed] [Google Scholar]
  12. Game J. C., Mortimer R. K. A genetic study of x-ray sensitive mutants in yeast. Mutat Res. 1974 Sep;24(3):281–292. doi: 10.1016/0027-5107(74)90176-6. [DOI] [PubMed] [Google Scholar]
  13. Garvik B., Carson M., Hartwell L. Single-stranded DNA arising at telomeres in cdc13 mutants may constitute a specific signal for the RAD9 checkpoint. Mol Cell Biol. 1995 Nov;15(11):6128–6138. doi: 10.1128/mcb.15.11.6128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gietz D., St Jean A., Woods R. A., Schiestl R. H. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 1992 Mar 25;20(6):1425–1425. doi: 10.1093/nar/20.6.1425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hartwell L. H., Kastan M. B. Cell cycle control and cancer. Science. 1994 Dec 16;266(5192):1821–1828. doi: 10.1126/science.7997877. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Holden D. W., Spanos A., Banks G. R. Nucleotide sequence of the REC1 gene of Ustilago maydis. Nucleic Acids Res. 1989 Dec 25;17(24):10489–10489. doi: 10.1093/nar/17.24.10489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Holliday R. Altered recombination frequencies in radiation sensitivie strains of Ustilago. Mutat Res. 1967 May-Jun;4(3):275–288. doi: 10.1016/0027-5107(67)90022-x. [DOI] [PubMed] [Google Scholar]
  19. Holliday R., Halliwell R. E., Evans M. W., Rowell V. Genetic characterization of rec-1, a mutant of Ustilago maydis defective in repair and recombination. Genet Res. 1976 Jun;27(3):413–453. doi: 10.1017/s0016672300016621. [DOI] [PubMed] [Google Scholar]
  20. Holliday R. Radiation sensitive mutants of Ustilago maydis. Mutat Res. 1965 Dec;2(6):557–559. doi: 10.1016/0027-5107(65)90022-9. [DOI] [PubMed] [Google Scholar]
  21. Holliday R., Taylor S. Y., Kmiec E. B., Holloman W. K. Biochemical characterization of rec1 mutants and the genetic control of recombination in Ustilago maydis. Cold Spring Harb Symp Quant Biol. 1984;49:669–673. doi: 10.1101/sqb.1984.049.01.075. [DOI] [PubMed] [Google Scholar]
  22. Johnson R. E., Kovvali G. K., Prakash L., Prakash S. Requirement of the yeast RTH1 5' to 3' exonuclease for the stability of simple repetitive DNA. Science. 1995 Jul 14;269(5221):238–240. doi: 10.1126/science.7618086. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Kanter-Smoler G., Knudsen K. E., Jimenez G., Sunnerhagen P., Subramani S. Separation of phenotypes in mutant alleles of the Schizosaccharomyces pombe cell-cycle checkpoint gene rad1+. Mol Biol Cell. 1995 Dec;6(12):1793–1805. doi: 10.1091/mbc.6.12.1793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lydall D., Weinert T. Yeast checkpoint genes in DNA damage processing: implications for repair and arrest. Science. 1995 Dec 1;270(5241):1488–1491. doi: 10.1126/science.270.5241.1488. [DOI] [PubMed] [Google Scholar]
  27. Marechal V., Elenbaas B., Piette J., Nicolas J. C., Levine A. J. The ribosomal L5 protein is associated with mdm-2 and mdm-2-p53 complexes. Mol Cell Biol. 1994 Nov;14(11):7414–7420. doi: 10.1128/mcb.14.11.7414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. McIntosh E. M. MCB elements and the regulation of DNA replication genes in yeast. Curr Genet. 1993 Sep;24(3):185–192. doi: 10.1007/BF00351790. [DOI] [PubMed] [Google Scholar]
  29. Morrison A., Johnson A. L., Johnston L. H., Sugino A. Pathway correcting DNA replication errors in Saccharomyces cerevisiae. EMBO J. 1993 Apr;12(4):1467–1473. doi: 10.1002/j.1460-2075.1993.tb05790.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Murray A. W. Creative blocks: cell-cycle checkpoints and feedback controls. Nature. 1992 Oct 15;359(6396):599–604. doi: 10.1038/359599a0. [DOI] [PubMed] [Google Scholar]
  31. Nelson W. G., Kastan M. B. DNA strand breaks: the DNA template alterations that trigger p53-dependent DNA damage response pathways. Mol Cell Biol. 1994 Mar;14(3):1815–1823. doi: 10.1128/mcb.14.3.1815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Onel K., Thelen M. P., Ferguson D. O., Bennett R. L., Holloman W. K. Mutation avoidance and DNA repair proficiency in Ustilago maydis are differentially lost with progressive truncation of the REC1 gene product. Mol Cell Biol. 1995 Oct;15(10):5329–5338. doi: 10.1128/mcb.15.10.5329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Reagan M. S., Pittenger C., Siede W., Friedberg E. C. Characterization of a mutant strain of Saccharomyces cerevisiae with a deletion of the RAD27 gene, a structural homolog of the RAD2 nucleotide excision repair gene. J Bacteriol. 1995 Jan;177(2):364–371. doi: 10.1128/jb.177.2.364-371.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Sandell L. L., Zakian V. A. Loss of a yeast telomere: arrest, recovery, and chromosome loss. Cell. 1993 Nov 19;75(4):729–739. doi: 10.1016/0092-8674(93)90493-a. [DOI] [PubMed] [Google Scholar]
  37. Savitsky K., Bar-Shira A., Gilad S., Rotman G., Ziv Y., Vanagaite L., Tagle D. A., Smith S., Uziel T., Sfez S. A single ataxia telangiectasia gene with a product similar to PI-3 kinase. Science. 1995 Jun 23;268(5218):1749–1753. doi: 10.1126/science.7792600. [DOI] [PubMed] [Google Scholar]
  38. Savitsky K., Sfez S., Tagle D. A., Ziv Y., Sartiel A., Collins F. S., Shiloh Y., Rotman G. The complete sequence of the coding region of the ATM gene reveals similarity to cell cycle regulators in different species. Hum Mol Genet. 1995 Nov;4(11):2025–2032. doi: 10.1093/hmg/4.11.2025. [DOI] [PubMed] [Google Scholar]
  39. Sheldrick K. S., Carr A. M. Feedback controls and G2 checkpoints: fission yeast as a model system. Bioessays. 1993 Dec;15(12):775–782. doi: 10.1002/bies.950151202. [DOI] [PubMed] [Google Scholar]
  40. Siede W. Cell cycle arrest in response to DNA damage: lessons from yeast. Mutat Res. 1995 Sep;337(2):73–84. doi: 10.1016/0921-8777(95)00023-d. [DOI] [PubMed] [Google Scholar]
  41. Siede W., Eckardt-Schupp F. DNA repair genes of Saccharomyces cerevisiae: complementing rad4 and rev2 mutations by plasmids which cannot be propagated in Escherichia coli. Curr Genet. 1986;11(3):205–210. doi: 10.1007/BF00420608. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. 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]
  44. Sunnerhagen P., Seaton B. L., Nasim A., Subramani S. Cloning and analysis of a gene involved in DNA repair and recombination, the rad1 gene of Schizosaccharomyces pombe. Mol Cell Biol. 1990 Jul;10(7):3750–3760. doi: 10.1128/mcb.10.7.3750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Thelen M. P., Onel K., Holloman W. K. The REC1 gene of Ustilago maydis involved in the cellular response to DNA damage encodes an exonuclease. J Biol Chem. 1994 Jan 7;269(1):747–754. [PubMed] [Google Scholar]
  46. 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]
  47. 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]
  48. 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]
  49. Weinert T., Lydall D. Cell cycle checkpoints, genetic instability and cancer. Semin Cancer Biol. 1993 Apr;4(2):129–140. [PubMed] [Google Scholar]
  50. Zheng P., Fay D. S., Burton J., Xiao H., Pinkham J. L., Stern D. F. SPK1 is an essential S-phase-specific gene of Saccharomyces cerevisiae that encodes a nuclear serine/threonine/tyrosine kinase. Mol Cell Biol. 1993 Sep;13(9):5829–5842. doi: 10.1128/mcb.13.9.5829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Zimmermann F. K. Sensitivity to methylmethanesulfonate and nitrous acid of ultraviolet light-sensitive mutants in Saccharomyces cerevisiae. Mol Gen Genet. 1968;102(3):247–256. doi: 10.1007/BF00385981. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES