Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1997 Jun;17(6):3037–3046. doi: 10.1128/mcb.17.6.3037

Reconstitution of a MEC1-independent checkpoint in yeast by expression of a novel human fork head cDNA.

D Pati 1, C Keller 1, M Groudine 1, S E Plon 1
PMCID: PMC232156  PMID: 9154802

Abstract

A novel human cDNA, CHES1 (checkpoint suppressor 1), has been isolated by suppression of the mec1-1 checkpoint mutation in Saccharomyces cerevisiae. CHES1 suppresses a number of DNA damage-activated checkpoint mutations in S. cerevisiae, including mec1, rad9, rad24, dun1, and rad53. CHES1 suppression of sensitivity to DNA damage is specific for checkpoint-defective strains, in contrast to DNA repair-defective strains. Presence of CHES1 but not a control vector resulted in G2 delay after UV irradiation in checkpoint-defective strains, with kinetics, nuclear morphology, and cycloheximide resistance similar to those of a wild-type strain. CHES1 can also suppress the lethality, UV sensitivity, and G2 checkpoint defect of a mec1 null mutation. In contrast to this activity, CHES1 had no measurable effect on the replication checkpoint as assayed by hydroxyurea sensitivity of a mec1 strain. Sequence analysis demonstrates that CHES1 is a novel member of the fork head/Winged Helix family of transcription factors. Suppression of the checkpoint-defective phenotype requires a 200-amino-acid domain in the carboxy terminus of the protein which is distinct from the DNA binding site. Analysis of CHES1 activity is most consistent with activation of an alternative MEC1-independent checkpoint pathway in budding yeast.

Full Text

The Full Text of this article is available as a PDF (1.5 MB).

Selected References

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

  1. Adams A. E., Pringle J. R. Relationship of actin and tubulin distribution to bud growth in wild-type and morphogenetic-mutant Saccharomyces cerevisiae. J Cell Biol. 1984 Mar;98(3):934–945. doi: 10.1083/jcb.98.3.934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  3. Baker T. A., Kremenstova E., Luo L. Complete transposition requires four active monomers in the mu transposase tetramer. Genes Dev. 1994 Oct 15;8(20):2416–2428. doi: 10.1101/gad.8.20.2416. [DOI] [PubMed] [Google Scholar]
  4. Burke D. J., Church D. Protein synthesis requirements for nuclear division, cytokinesis, and cell separation in Saccharomyces cerevisiae. Mol Cell Biol. 1991 Jul;11(7):3691–3698. doi: 10.1128/mcb.11.7.3691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carr A. M. Checkpoints take the next step. Science. 1996 Jan 19;271(5247):314–315. doi: 10.1126/science.271.5247.314. [DOI] [PubMed] [Google Scholar]
  6. Colicelli J., Birchmeier C., Michaeli T., O'Neill K., Riggs M., Wigler M. Isolation and characterization of a mammalian gene encoding a high-affinity cAMP phosphodiesterase. Proc Natl Acad Sci U S A. 1989 May;86(10):3599–3603. doi: 10.1073/pnas.86.10.3599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Colicelli J., Nicolette C., Birchmeier C., Rodgers L., Riggs M., Wigler M. Expression of three mammalian cDNAs that interfere with RAS function in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1991 Apr 1;88(7):2913–2917. doi: 10.1073/pnas.88.7.2913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Davey S., Beach D. RACH2, a novel human gene that complements a fission yeast cell cycle checkpoint mutation. Mol Biol Cell. 1995 Oct;6(10):1411–1421. doi: 10.1091/mbc.6.10.1411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Elledge S. J. Cell cycle checkpoints: preventing an identity crisis. Science. 1996 Dec 6;274(5293):1664–1672. doi: 10.1126/science.274.5293.1664. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Field L. L., Tobias R., Thomson G., Plon S. Susceptibility to insulin-dependent diabetes mellitus maps to a locus (IDDM11) on human chromosome 14q24.3-q31. Genomics. 1996 Apr 1;33(1):1–8. doi: 10.1006/geno.1996.0153. [DOI] [PubMed] [Google Scholar]
  13. Ford J. C., al-Khodairy F., Fotou E., Sheldrick K. S., Griffiths D. J., Carr A. M. 14-3-3 protein homologs required for the DNA damage checkpoint in fission yeast. Science. 1994 Jul 22;265(5171):533–535. doi: 10.1126/science.8036497. [DOI] [PubMed] [Google Scholar]
  14. Fornace A. J., Jr Mammalian genes induced by radiation; activation of genes associated with growth control. Annu Rev Genet. 1992;26:507–526. doi: 10.1146/annurev.ge.26.120192.002451. [DOI] [PubMed] [Google Scholar]
  15. Galili N., Davis R. J., Fredericks W. J., Mukhopadhyay S., Rauscher F. J., 3rd, Emanuel B. S., Rovera G., Barr F. G. Fusion of a fork head domain gene to PAX3 in the solid tumour alveolar rhabdomyosarcoma. Nat Genet. 1993 Nov;5(3):230–235. doi: 10.1038/ng1193-230. [DOI] [PubMed] [Google Scholar]
  16. Greenwell P. W., Kronmal S. L., Porter S. E., Gassenhuber J., Obermaier B., Petes T. D. TEL1, a gene involved in controlling telomere length in S. cerevisiae, is homologous to the human ataxia telangiectasia gene. Cell. 1995 Sep 8;82(5):823–829. doi: 10.1016/0092-8674(95)90479-4. [DOI] [PubMed] [Google Scholar]
  17. Hari K. L., Santerre A., Sekelsky J. J., McKim K. S., Boyd J. B., Hawley R. S. The mei-41 gene of D. melanogaster is a structural and functional homolog of the human ataxia telangiectasia gene. Cell. 1995 Sep 8;82(5):815–821. doi: 10.1016/0092-8674(95)90478-6. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Hawley R. S., Friend S. H. Strange bedfellows in even stranger places: the role of ATM in meiotic cells, lymphocytes, tumors, and its functional links to p53. Genes Dev. 1996 Oct 1;10(19):2383–2388. doi: 10.1101/gad.10.19.2383. [DOI] [PubMed] [Google Scholar]
  20. Hoffman C. S., Winston F. A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene. 1987;57(2-3):267–272. doi: 10.1016/0378-1119(87)90131-4. [DOI] [PubMed] [Google Scholar]
  21. Hofmann K., Bucher P. The FHA domain: a putative nuclear signalling domain found in protein kinases and transcription factors. Trends Biochem Sci. 1995 Sep;20(9):347–349. doi: 10.1016/s0968-0004(00)89072-6. [DOI] [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. Jimenez G., Yucel J., Rowley R., Subramani S. The rad3+ gene of Schizosaccharomyces pombe is involved in multiple checkpoint functions and in DNA repair. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):4952–4956. doi: 10.1073/pnas.89.11.4952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kato R., Ogawa H. An essential gene, ESR1, is required for mitotic cell growth, DNA repair and meiotic recombination in Saccharomyces cerevisiae. Nucleic Acids Res. 1994 Aug 11;22(15):3104–3112. doi: 10.1093/nar/22.15.3104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kiser G. L., Weinert T. A. Distinct roles of yeast MEC and RAD checkpoint genes in transcriptional induction after DNA damage and implications for function. Mol Biol Cell. 1996 May;7(5):703–718. doi: 10.1091/mbc.7.5.703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Lai E., Prezioso V. R., Tao W. F., Chen W. S., Darnell J. E., Jr Hepatocyte nuclear factor 3 alpha belongs to a gene family in mammals that is homologous to the Drosophila homeotic gene fork head. Genes Dev. 1991 Mar;5(3):416–427. doi: 10.1101/gad.5.3.416. [DOI] [PubMed] [Google Scholar]
  28. Li C., Lusis A. J., Sparkes R., Tran S. M., Gaynor R. Characterization and chromosomal mapping of the gene encoding the cellular DNA binding protein HTLF. Genomics. 1992 Jul;13(3):658–664. doi: 10.1016/0888-7543(92)90138-i. [DOI] [PubMed] [Google Scholar]
  29. Lieberman H. B., Hopkins K. M., Nass M., Demetrick D., Davey S. A human homolog of the Schizosaccharomyces pombe rad9+ checkpoint control gene. Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):13890–13895. doi: 10.1073/pnas.93.24.13890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lundblad V., Blackburn E. H. An alternative pathway for yeast telomere maintenance rescues est1- senescence. Cell. 1993 Apr 23;73(2):347–360. doi: 10.1016/0092-8674(93)90234-h. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Morrow D. M., Tagle D. A., Shiloh Y., Collins F. S., Hieter P. TEL1, an S. cerevisiae homolog of the human gene mutated in ataxia telangiectasia, is functionally related to the yeast checkpoint gene MEC1. Cell. 1995 Sep 8;82(5):831–840. doi: 10.1016/0092-8674(95)90480-8. [DOI] [PubMed] [Google Scholar]
  33. Murakami H., Okayama H. A kinase from fission yeast responsible for blocking mitosis in S phase. Nature. 1995 Apr 27;374(6525):817–819. doi: 10.1038/374817a0. [DOI] [PubMed] [Google Scholar]
  34. Navas T. A., Zhou Z., Elledge S. J. DNA polymerase epsilon links the DNA replication machinery to the S phase checkpoint. Cell. 1995 Jan 13;80(1):29–39. doi: 10.1016/0092-8674(95)90448-4. [DOI] [PubMed] [Google Scholar]
  35. Nehls M., Pfeifer D., Schorpp M., Hedrich H., Boehm T. New member of the winged-helix protein family disrupted in mouse and rat nude mutations. Nature. 1994 Nov 3;372(6501):103–107. doi: 10.1038/372103a0. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Plon S. E., Leppig K. A., Do H. N., Groudine M. Cloning of the human homolog of the CDC34 cell cycle gene by complementation in yeast. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10484–10488. doi: 10.1073/pnas.90.22.10484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Prakash S., Sung P., Prakash L. DNA repair genes and proteins of Saccharomyces cerevisiae. Annu Rev Genet. 1993;27:33–70. doi: 10.1146/annurev.ge.27.120193.000341. [DOI] [PubMed] [Google Scholar]
  39. Rose M., Botstein D. Construction and use of gene fusions to lacZ (beta-galactosidase) that are expressed in yeast. Methods Enzymol. 1983;101:167–180. doi: 10.1016/0076-6879(83)01012-5. [DOI] [PubMed] [Google Scholar]
  40. Rowley R., Subramani S., Young P. G. Checkpoint controls in Schizosaccharomyces pombe: rad1. EMBO J. 1992 Apr;11(4):1335–1342. doi: 10.1002/j.1460-2075.1992.tb05178.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sanchez Y., Desany B. A., Jones W. J., Liu Q., Wang B., Elledge S. J. Regulation of RAD53 by the ATM-like kinases MEC1 and TEL1 in yeast cell cycle checkpoint pathways. Science. 1996 Jan 19;271(5247):357–360. doi: 10.1126/science.271.5247.357. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. Schiestl R. H., Gietz R. D. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet. 1989 Dec;16(5-6):339–346. doi: 10.1007/BF00340712. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. Seaton B. L., Yucel J., Sunnerhagen P., Subramani S. Isolation and characterization of the Schizosaccharomyces pombe rad3 gene, involved in the DNA damage and DNA synthesis checkpoints. Gene. 1992 Sep 21;119(1):83–89. doi: 10.1016/0378-1119(92)90069-2. [DOI] [PubMed] [Google Scholar]
  46. Shapiro D. N., Sublett J. E., Li B., Downing J. R., Naeve C. W. Fusion of PAX3 to a member of the forkhead family of transcription factors in human alveolar rhabdomyosarcoma. Cancer Res. 1993 Nov 1;53(21):5108–5112. [PubMed] [Google Scholar]
  47. 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]
  48. Smith R. F., Smith T. F. Automatic generation of primary sequence patterns from sets of related protein sequences. Proc Natl Acad Sci U S A. 1990 Jan;87(1):118–122. doi: 10.1073/pnas.87.1.118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Stern D. F., Zheng P., Beidler D. R., Zerillo C. Spk1, a new kinase from Saccharomyces cerevisiae, phosphorylates proteins on serine, threonine, and tyrosine. Mol Cell Biol. 1991 Feb;11(2):987–1001. doi: 10.1128/mcb.11.2.987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Thompson J. D., Higgins D. G., Gibson T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994 Nov 11;22(22):4673–4680. doi: 10.1093/nar/22.22.4673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Walworth N., Davey S., Beach D. Fission yeast chk1 protein kinase links the rad checkpoint pathway to cdc2. Nature. 1993 May 27;363(6427):368–371. doi: 10.1038/363368a0. [DOI] [PubMed] [Google Scholar]
  52. Weigel D., Jäckle H. The fork head domain: a novel DNA binding motif of eukaryotic transcription factors? Cell. 1990 Nov 2;63(3):455–456. doi: 10.1016/0092-8674(90)90439-l. [DOI] [PubMed] [Google Scholar]
  53. 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]
  54. 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]
  55. 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]
  56. 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]
  57. Weinert T., Lydall D. Cell cycle checkpoints, genetic instability and cancer. Semin Cancer Biol. 1993 Apr;4(2):129–140. [PubMed] [Google Scholar]
  58. Yan Y. X., Schiestl R. H., Prakash L. Mating-type suppression of the DNA-repair defect of the yeast rad6 delta mutation requires the activity of genes in the RAD52 epistasis group. Curr Genet. 1995 Jun;28(1):12–18. doi: 10.1007/BF00311876. [DOI] [PubMed] [Google Scholar]
  59. 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]
  60. al-Khodairy F., Carr A. M. DNA repair mutants defining G2 checkpoint pathways in Schizosaccharomyces pombe. EMBO J. 1992 Apr;11(4):1343–1350. doi: 10.1002/j.1460-2075.1992.tb05179.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. al-Khodairy F., Fotou E., Sheldrick K. S., Griffiths D. J., Lehmann A. R., Carr A. M. Identification and characterization of new elements involved in checkpoint and feedback controls in fission yeast. Mol Biol Cell. 1994 Feb;5(2):147–160. doi: 10.1091/mbc.5.2.147. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES