Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1990 Dec;10(12):6554–6564. doi: 10.1128/mcb.10.12.6554

Characterization of RAD9 of Saccharomyces cerevisiae and evidence that its function acts posttranslationally in cell cycle arrest after DNA damage.

T A Weinert 1, L H Hartwell 1
PMCID: PMC362932  PMID: 2247073

Abstract

In eucaryotic cells, incompletely replicated or damaged chromosomes induce cell cycle arrest in G2 before mitosis, and in the yeast Saccharomyces cerevisiae the RAD9 gene is essential for the cell cycle arrest (T.A. Weinert and L. H. Hartwell, Science 241:317-322, 1988). In this report, we extend the analysis of RAD9-dependent cell cycle control. We found that both induction of RAD9-dependent arrest in G2 and recovery from arrest could occur in the presence of the protein synthesis inhibitor cycloheximide, showing that the mechanism of RAD9-dependent control involves a posttranslational mechanism(s). We have isolated and determined the DNA sequence of the RAD9 gene, confirming the DNA sequence reported previously (R. H. Schiestl, P. Reynolds, S. Prakash, and L. Prakash, Mol. Cell. Biol. 9:1882-1886, 1989). The predicted protein sequence for the Rad9 protein bears no similarity to sequences of known proteins. We also found that synthesis of the RAD9 transcript in the cell cycle was constitutive and not induced by X-irradiation. We constructed yeast cells containing a complete deletion of the RAD9 gene; the rad9 null mutants were viable, sensitive to X- and UV irradiation, and defective for cell cycle arrest after DNA damage. Although Rad+ and rad9 delta cells had similar growth rates and cell cycle kinetics in unirradiated cells, the spontaneous rate of chromosome loss (in unirradiated cells) was elevated 7- to 21-fold in rad9 delta cells. These studies show that in the presence of induced or endogenous DNA damage, RAD9 is a negative regulator that inhibits progression from G2 in order to preserve cell viability and to maintain the fidelity of chromosome transmission.

Full text

PDF
6554

Images in this article

6557Image
on p.6557
6557Image
on p.6557
6559Image
on p.6559

Selected References

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

  1. BURNS V. W. X-ray-induced division delay of individual yeast cells. Radiat Res. 1956 May;4(5):394–412. [PubMed] [Google Scholar]
  2. Beach D., Durkacz B., Nurse P. Functionally homologous cell cycle control genes in budding and fission yeast. Nature. 1982 Dec 23;300(5894):706–709. doi: 10.1038/300706a0. [DOI] [PubMed] [Google Scholar]
  3. Brunborg G., Resnick M. A., Williamson D. H. Cell-cycle-specific repair of DNA double strand breaks in Saccharomyces cerevisiae. Radiat Res. 1980 Jun;82(3):547–558. [PubMed] [Google Scholar]
  4. Brunborg G., Williamson D. H. The relevance of the nuclear division cycle to radiosensitivity in yeast. Mol Gen Genet. 1978 Jul 4;162(3):277–286. doi: 10.1007/BF00268853. [DOI] [PubMed] [Google Scholar]
  5. Busse P. M., Bose S. K., Jones R. W., Tolmach L. J. The action of caffeine on X-irradiated HeLa cells. III. Enhancement of X-ray-induced killing during G2 arrest. Radiat Res. 1978 Nov;76(2):292–307. [PubMed] [Google Scholar]
  6. Carson M. J., Hartwell L. CDC17: an essential gene that prevents telomere elongation in yeast. Cell. 1985 Aug;42(1):249–257. doi: 10.1016/s0092-8674(85)80120-3. [DOI] [PubMed] [Google Scholar]
  7. Cole G. M., Schild D., Lovett S. T., Mortimer R. K. Regulation of RAD54- and RAD52-lacZ gene fusions in Saccharomyces cerevisiae in response to DNA damage. Mol Cell Biol. 1987 Mar;7(3):1078–1084. doi: 10.1128/mcb.7.3.1078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dowling E. L., Maloney D. H., Fogel S. Meiotic recombination and sporulation in repair-deficient strains of yeast. Genetics. 1985 Feb;109(2):283–302. doi: 10.1093/genetics/109.2.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Elledge S. J., Davis R. W. Identification and isolation of the gene encoding the small subunit of ribonucleotide reductase from Saccharomyces cerevisiae: DNA damage-inducible gene required for mitotic viability. Mol Cell Biol. 1987 Aug;7(8):2783–2793. doi: 10.1128/mcb.7.8.2783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Enoch T., Nurse P. Mutation of fission yeast cell cycle control genes abolishes dependence of mitosis on DNA replication. Cell. 1990 Feb 23;60(4):665–673. doi: 10.1016/0092-8674(90)90669-6. [DOI] [PubMed] [Google Scholar]
  11. Fantes P. A. Dependency relations between events in mitosis in Schizosaccharomyces pombe. J Cell Sci. 1982 Jun;55:383–402. doi: 10.1242/jcs.55.1.383. [DOI] [PubMed] [Google Scholar]
  12. Gould K. L., Nurse P. Tyrosine phosphorylation of the fission yeast cdc2+ protein kinase regulates entry into mitosis. Nature. 1989 Nov 2;342(6245):39–45. doi: 10.1038/342039a0. [DOI] [PubMed] [Google Scholar]
  13. Harnden D. G., Taylor A. M. Chromosomes and neoplasia. Adv Hum Genet. 1979;9:1-70, 355-60. doi: 10.1007/978-1-4615-8276-2_1. [DOI] [PubMed] [Google Scholar]
  14. Hartshorne T. A., Blumberg H., Young E. T. Sequence homology of the yeast regulatory protein ADR1 with Xenopus transcription factor TFIIIA. Nature. 1986 Mar 20;320(6059):283–287. doi: 10.1038/320283a0. [DOI] [PubMed] [Google Scholar]
  15. Hartwell L. H. Macromolecule synthesis in temperature-sensitive mutants of yeast. J Bacteriol. 1967 May;93(5):1662–1670. doi: 10.1128/jb.93.5.1662-1670.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hartwell L. H., Smith D. Altered fidelity of mitotic chromosome transmission in cell cycle mutants of S. cerevisiae. Genetics. 1985 Jul;110(3):381–395. doi: 10.1093/genetics/110.3.381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Henikoff S. Unidirectional digestion with exonuclease III in DNA sequence analysis. Methods Enzymol. 1987;155:156–165. doi: 10.1016/0076-6879(87)55014-5. [DOI] [PubMed] [Google Scholar]
  19. Hereford L. M., Osley M. A., Ludwig T. R., 2nd, McLaughlin C. S. Cell-cycle regulation of yeast histone mRNA. Cell. 1981 May;24(2):367–375. doi: 10.1016/0092-8674(81)90326-3. [DOI] [PubMed] [Google Scholar]
  20. Hindley J., Phear G. A. Sequence of the cell division gene CDC2 from Schizosaccharomyces pombe; patterns of splicing and homology to protein kinases. Gene. 1984 Nov;31(1-3):129–134. doi: 10.1016/0378-1119(84)90203-8. [DOI] [PubMed] [Google Scholar]
  21. Hinnen A., Hicks J. B., Fink G. R. Transformation of yeast. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1929–1933. doi: 10.1073/pnas.75.4.1929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jacobs C. W., Adams A. E., Szaniszlo P. J., Pringle J. R. Functions of microtubules in the Saccharomyces cerevisiae cell cycle. J Cell Biol. 1988 Oct;107(4):1409–1426. doi: 10.1083/jcb.107.4.1409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lau C. C., Pardee A. B. Mechanism by which caffeine potentiates lethality of nitrogen mustard. Proc Natl Acad Sci U S A. 1982 May;79(9):2942–2946. doi: 10.1073/pnas.79.9.2942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Little J. W., Mount D. W. The SOS regulatory system of Escherichia coli. Cell. 1982 May;29(1):11–22. doi: 10.1016/0092-8674(82)90085-x. [DOI] [PubMed] [Google Scholar]
  26. Lörincz A. T., Reed S. I. Primary structure homology between the product of yeast cell division control gene CDC28 and vertebrate oncogenes. Nature. 1984 Jan 12;307(5947):183–185. doi: 10.1038/307183a0. [DOI] [PubMed] [Google Scholar]
  27. McClanahan T., McEntee K. Specific transcripts are elevated in Saccharomyces cerevisiae in response to DNA damage. Mol Cell Biol. 1984 Nov;4(11):2356–2363. doi: 10.1128/mcb.4.11.2356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mortimer R. K., Schild D. Genetic map of Saccharomyces cerevisiae, edition 9. Microbiol Rev. 1985 Sep;49(3):181–213. doi: 10.1128/mr.49.3.181-213.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Murray A. W., Kirschner M. W. Cyclin synthesis drives the early embryonic cell cycle. Nature. 1989 May 25;339(6222):275–280. doi: 10.1038/339275a0. [DOI] [PubMed] [Google Scholar]
  30. Murray A. W., Kirschner M. W. Dominoes and clocks: the union of two views of the cell cycle. Science. 1989 Nov 3;246(4930):614–621. doi: 10.1126/science.2683077. [DOI] [PubMed] [Google Scholar]
  31. Nishimoto T., Eilen E., Basilico C. Premature of chromosome condensation in a ts DNA- mutant of BHK cells. Cell. 1978 Oct;15(2):475–483. doi: 10.1016/0092-8674(78)90017-x. [DOI] [PubMed] [Google Scholar]
  32. Nurse P. Universal control mechanism regulating onset of M-phase. Nature. 1990 Apr 5;344(6266):503–508. doi: 10.1038/344503a0. [DOI] [PubMed] [Google Scholar]
  33. Osmani S. A., Engle D. B., Doonan J. H., Morris N. R. Spindle formation and chromatin condensation in cells blocked at interphase by mutation of a negative cell cycle control gene. Cell. 1988 Jan 29;52(2):241–251. doi: 10.1016/0092-8674(88)90513-2. [DOI] [PubMed] [Google Scholar]
  34. Piggott J. R., Rai R., Carter B. L. A bifunctional gene product involved in two phases of the yeast cell cycle. Nature. 1982 Jul 22;298(5872):391–393. doi: 10.1038/298391a0. [DOI] [PubMed] [Google Scholar]
  35. Quinlan R. A., Pogson C. I., Gull K. The influence of the microtubule inhibitor, methyl benzimidazol-2-yl-carbamate (MBC) on nuclear division and the cell cycle in Saccharomyces cerevisiae. J Cell Sci. 1980 Dec;46:341–352. doi: 10.1242/jcs.46.1.341. [DOI] [PubMed] [Google Scholar]
  36. Rose M. D., Fink G. R. KAR1, a gene required for function of both intranuclear and extranuclear microtubules in yeast. Cell. 1987 Mar 27;48(6):1047–1060. doi: 10.1016/0092-8674(87)90712-4. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Rowley R. Is G2-arrest an active cellular response to irradiation? Int J Radiat Biol Relat Stud Phys Chem Med. 1985 Nov;48(5):811–820. doi: 10.1080/09553008514551911. [DOI] [PubMed] [Google Scholar]
  39. Ruby S. W., Szostak J. W. Specific Saccharomyces cerevisiae genes are expressed in response to DNA-damaging agents. Mol Cell Biol. 1985 Jan;5(1):75–84. doi: 10.1128/mcb.5.1.75. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Russell P., Moreno S., Reed S. I. Conservation of mitotic controls in fission and budding yeasts. Cell. 1989 Apr 21;57(2):295–303. doi: 10.1016/0092-8674(89)90967-7. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Schlegel R., Pardee A. B. Caffeine-induced uncoupling of mitosis from the completion of DNA replication in mammalian cells. Science. 1986 Jun 6;232(4755):1264–1266. doi: 10.1126/science.2422760. [DOI] [PubMed] [Google Scholar]
  43. Sunkara P. S., Wright D. A., Rao P. N. Mitotic factors from mammalian cells induce germinal vesicle breakdown and chromosome condensation in amphibian oocytes. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2799–2802. doi: 10.1073/pnas.76.6.2799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Tolmach L. J., Jones R. W., Busse P. M. The action of caffeine on X-irradiated HeLa cells. I. Delayed inhibition of DNA synthesis. Radiat Res. 1977 Sep;71(3):653–665. [PubMed] [Google Scholar]
  45. Treger J. M., Heichman K. A., McEntee K. Expression of the yeast UB14 gene increases in response to DNA-damaging agents and in meiosis. Mol Cell Biol. 1988 Mar;8(3):1132–1136. doi: 10.1128/mcb.8.3.1132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Walters R. A., Petersen D. F. Radiosensivity of mammalian cells. I. Timing and dose-dependence of radiation-induced division delay. Biophys J. 1968 Dec;8(12):1475–1486. doi: 10.1016/s0006-3495(68)86567-1. [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. Williamson D. H., Fennell D. J. The use of fluorescent DNA-binding agent for detecting and separating yeast mitochondrial DNA. Methods Cell Biol. 1975;12:335–351. doi: 10.1016/s0091-679x(08)60963-2. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES