Skip to main content
PMC home page
Genetics logoLink to Genetics
. 2003 Dec;165(4):1717–1732. doi: 10.1093/genetics/165.4.1717

Functions of Saccharomyces cerevisiae 14-3-3 proteins in response to DNA damage and to DNA replication stress.

Francisca Lottersberger 1, Fabio Rubert 1, Veronica Baldo 1, Giovanna Lucchini 1, Maria Pia Longhese 1
PMCID: PMC1462906  PMID: 14704161

Abstract

Two members of the 14-3-3 protein family, involved in key biological processes in different eukaryotes, are encoded by the functionally redundant Saccharomyces cerevisiae BMH1 and BMH2 genes. We produced and characterized 12 independent bmh1 mutant alleles, whose presence in the cell as the sole 14-3-3 source causes hypersensitivity to genotoxic agents, indicating that Bmh proteins are required for proper response to DNA damage. In particular, the bmh1-103 and bmh1-266 mutant alleles cause defects in G1/S and G2/M DNA damage checkpoints, whereas only the G2/M checkpoint is altered by the bmh1-169 and bmh1-221 alleles. Impaired checkpoint responses correlate with the inability to maintain phosphorylated forms of Rad53 and/or Chk1, suggesting that Bmh proteins might regulate phosphorylation/dephosphorylation of these checkpoint kinases. Moreover, several bmh1 bmh2Delta mutants are defective in resuming DNA replication after transient deoxynucleotide depletion, and all display synthetic effects when also carrying mutations affecting the polalpha-primase and RPA DNA replication complexes, suggesting a role for Bmh proteins in DNA replication stress response. Finally, the bmh1-169 bmh2Delta and bmh1-170 bmh2Delta mutants show increased rates of spontaneous gross chromosomal rearrangements, indicating that Bmh proteins are required to suppress genome instability.

Full Text

The Full Text of this article is available as a PDF (765.5 KB).

Selected References

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

  1. Bentley N. J., Holtzman D. A., Flaggs G., Keegan K. S., DeMaggio A., Ford J. C., Hoekstra M., Carr A. M. The Schizosaccharomyces pombe rad3 checkpoint gene. EMBO J. 1996 Dec 2;15(23):6641–6651. [PMC free article] [PubMed] [Google Scholar]
  2. Blasina A., de Weyer I. V., Laus M. C., Luyten W. H., Parker A. E., McGowan C. H. A human homologue of the checkpoint kinase Cds1 directly inhibits Cdc25 phosphatase. Curr Biol. 1999 Jan 14;9(1):1–10. doi: 10.1016/s0960-9822(99)80041-4. [DOI] [PubMed] [Google Scholar]
  3. Chan T. A., Hermeking H., Lengauer C., Kinzler K. W., Vogelstein B. 14-3-3Sigma is required to prevent mitotic catastrophe after DNA damage. Nature. 1999 Oct 7;401(6753):616–620. doi: 10.1038/44188. [DOI] [PubMed] [Google Scholar]
  4. Chiang C. W., Harris G., Ellig C., Masters S. C., Subramanian R., Shenolikar S., Wadzinski B. E., Yang E. Protein phosphatase 2A activates the proapoptotic function of BAD in interleukin- 3-dependent lymphoid cells by a mechanism requiring 14-3-3 dissociation. Blood. 2001 Mar 1;97(5):1289–1297. doi: 10.1182/blood.v97.5.1289. [DOI] [PubMed] [Google Scholar]
  5. Cocker J. H., Piatti S., Santocanale C., Nasmyth K., Diffley J. F. An essential role for the Cdc6 protein in forming the pre-replicative complexes of budding yeast. Nature. 1996 Jan 11;379(6561):180–182. doi: 10.1038/379180a0. [DOI] [PubMed] [Google Scholar]
  6. Cortez D., Guntuku S., Qin J., Elledge S. J. ATR and ATRIP: partners in checkpoint signaling. Science. 2001 Nov 23;294(5547):1713–1716. doi: 10.1126/science.1065521. [DOI] [PubMed] [Google Scholar]
  7. Cvrcková F., Nasmyth K. Yeast G1 cyclins CLN1 and CLN2 and a GAP-like protein have a role in bud formation. EMBO J. 1993 Dec 15;12(13):5277–5286. doi: 10.1002/j.1460-2075.1993.tb06223.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dalal S. N., Schweitzer C. M., Gan J., DeCaprio J. A. Cytoplasmic localization of human cdc25C during interphase requires an intact 14-3-3 binding site. Mol Cell Biol. 1999 Jun;19(6):4465–4479. doi: 10.1128/mcb.19.6.4465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Desany B. A., Alcasabas A. A., Bachant J. B., Elledge S. J. Recovery from DNA replicational stress is the essential function of the S-phase checkpoint pathway. Genes Dev. 1998 Sep 15;12(18):2956–2970. doi: 10.1101/gad.12.18.2956. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dhar S., Squire J. A., Hande M. P., Wellinger R. J., Pandita T. K. Inactivation of 14-3-3sigma influences telomere behavior and ionizing radiation-induced chromosomal instability. Mol Cell Biol. 2000 Oct;20(20):7764–7772. doi: 10.1128/mcb.20.20.7764-7772.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Edwards R. J., Bentley N. J., Carr A. M. A Rad3-Rad26 complex responds to DNA damage independently of other checkpoint proteins. Nat Cell Biol. 1999 Nov;1(7):393–398. doi: 10.1038/15623. [DOI] [PubMed] [Google Scholar]
  12. Emili A. MEC1-dependent phosphorylation of Rad9p in response to DNA damage. Mol Cell. 1998 Aug;2(2):183–189. doi: 10.1016/s1097-2765(00)80128-8. [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. Fu H., Subramanian R. R., Masters S. C. 14-3-3 proteins: structure, function, and regulation. Annu Rev Pharmacol Toxicol. 2000;40:617–647. doi: 10.1146/annurev.pharmtox.40.1.617. [DOI] [PubMed] [Google Scholar]
  15. Furnari B., Rhind N., Russell P. Cdc25 mitotic inducer targeted by chk1 DNA damage checkpoint kinase. Science. 1997 Sep 5;277(5331):1495–1497. doi: 10.1126/science.277.5331.1495. [DOI] [PubMed] [Google Scholar]
  16. Gelperin D., Weigle J., Nelson K., Roseboom P., Irie K., Matsumoto K., Lemmon S. 14-3-3 proteins: potential roles in vesicular transport and Ras signaling in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1995 Dec 5;92(25):11539–11543. doi: 10.1073/pnas.92.25.11539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gietz R. D., Sugino A. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene. 1988 Dec 30;74(2):527–534. doi: 10.1016/0378-1119(88)90185-0. [DOI] [PubMed] [Google Scholar]
  18. Gilbert C. S., Green C. M., Lowndes N. F. Budding yeast Rad9 is an ATP-dependent Rad53 activating machine. Mol Cell. 2001 Jul;8(1):129–136. doi: 10.1016/s1097-2765(01)00267-2. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Hartwell L. H. Genetic control of the cell division cycle in yeast. II. Genes controlling DNA replication and its initiation. J Mol Biol. 1971 Jul 14;59(1):183–194. doi: 10.1016/0022-2836(71)90420-7. [DOI] [PubMed] [Google Scholar]
  22. Holt R. D., Chidiac R. H., Rule D. C. Dental treatment for children under general anaesthesia in day care facilities at a London dental hospital. Br Dent J. 1991 Apr 6;170(7):262–266. doi: 10.1038/sj.bdj.4807504. [DOI] [PubMed] [Google Scholar]
  23. Jelinek T., Dent P., Sturgill T. W., Weber M. J. Ras-induced activation of Raf-1 is dependent on tyrosine phosphorylation. Mol Cell Biol. 1996 Mar;16(3):1027–1034. doi: 10.1128/mcb.16.3.1027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kumagai A., Dunphy W. G. Binding of 14-3-3 proteins and nuclear export control the intracellular localization of the mitotic inducer Cdc25. Genes Dev. 1999 May 1;13(9):1067–1072. doi: 10.1101/gad.13.9.1067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lee J., Kumagai A., Dunphy W. G. Positive regulation of Wee1 by Chk1 and 14-3-3 proteins. Mol Biol Cell. 2001 Mar;12(3):551–563. doi: 10.1091/mbc.12.3.551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Longhese M. P., Foiani M., Muzi-Falconi M., Lucchini G., Plevani P. DNA damage checkpoint in budding yeast. EMBO J. 1998 Oct 1;17(19):5525–5528. doi: 10.1093/emboj/17.19.5525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Longhese M. P., Fraschini R., Plevani P., Lucchini G. Yeast pip3/mec3 mutants fail to delay entry into S phase and to slow DNA replication in response to DNA damage, and they define a functional link between Mec3 and DNA primase. Mol Cell Biol. 1996 Jul;16(7):3235–3244. doi: 10.1128/mcb.16.7.3235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Longhese M. P., Jovine L., Plevani P., Lucchini G. Conditional mutations in the yeast DNA primase genes affect different aspects of DNA metabolism and interactions in the DNA polymerase alpha-primase complex. Genetics. 1993 Feb;133(2):183–191. doi: 10.1093/genetics/133.2.183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Longhese M. P., Paciotti V., Fraschini R., Zaccarini R., Plevani P., Lucchini G. The novel DNA damage checkpoint protein ddc1p is phosphorylated periodically during the cell cycle and in response to DNA damage in budding yeast. EMBO J. 1997 Sep 1;16(17):5216–5226. doi: 10.1093/emboj/16.17.5216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Longhese M. P., Plevani P., Lucchini G. Replication factor A is required in vivo for DNA replication, repair, and recombination. Mol Cell Biol. 1994 Dec;14(12):7884–7890. doi: 10.1128/mcb.14.12.7884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lopes M., Cotta-Ramusino C., Pellicioli A., Liberi G., Plevani P., Muzi-Falconi M., Newlon C. S., Foiani M. The DNA replication checkpoint response stabilizes stalled replication forks. Nature. 2001 Aug 2;412(6846):557–561. doi: 10.1038/35087613. [DOI] [PubMed] [Google Scholar]
  32. Lopez-Girona A., Kanoh J., Russell P. Nuclear exclusion of Cdc25 is not required for the DNA damage checkpoint in fission yeast. Curr Biol. 2001 Jan 9;11(1):50–54. doi: 10.1016/s0960-9822(00)00026-9. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Moser B. A., Russell P. Cell cycle regulation in Schizosaccharomyces pombe. Curr Opin Microbiol. 2000 Dec;3(6):631–636. doi: 10.1016/s1369-5274(00)00152-1. [DOI] [PubMed] [Google Scholar]
  35. Myung K., Datta A., Kolodner R. D. Suppression of spontaneous chromosomal rearrangements by S phase checkpoint functions in Saccharomyces cerevisiae. Cell. 2001 Feb 9;104(3):397–408. doi: 10.1016/s0092-8674(01)00227-6. [DOI] [PubMed] [Google Scholar]
  36. Neecke H., Lucchini G., Longhese M. P. Cell cycle progression in the presence of irreparable DNA damage is controlled by a Mec1- and Rad53-dependent checkpoint in budding yeast. EMBO J. 1999 Aug 16;18(16):4485–4497. doi: 10.1093/emboj/18.16.4485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Paciotti V., Clerici M., Lucchini G., Longhese M. P. The checkpoint protein Ddc2, functionally related to S. pombe Rad26, interacts with Mec1 and is regulated by Mec1-dependent phosphorylation in budding yeast. Genes Dev. 2000 Aug 15;14(16):2046–2059. [PMC free article] [PubMed] [Google Scholar]
  38. Peng C. Y., Graves P. R., Thoma R. S., Wu Z., Shaw A. S., Piwnica-Worms H. Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by phosphorylation of Cdc25C on serine-216. Science. 1997 Sep 5;277(5331):1501–1505. doi: 10.1126/science.277.5331.1501. [DOI] [PubMed] [Google Scholar]
  39. Piatti S., Lengauer C., Nasmyth K. Cdc6 is an unstable protein whose de novo synthesis in G1 is important for the onset of S phase and for preventing a 'reductional' anaphase in the budding yeast Saccharomyces cerevisiae. EMBO J. 1995 Aug 1;14(15):3788–3799. doi: 10.1002/j.1460-2075.1995.tb00048.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Pizzagalli A., Valsasnini P., Plevani P., Lucchini G. DNA polymerase I gene of Saccharomyces cerevisiae: nucleotide sequence, mapping of a temperature-sensitive mutation, and protein homology with other DNA polymerases. Proc Natl Acad Sci U S A. 1988 Jun;85(11):3772–3776. doi: 10.1073/pnas.85.11.3772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Roberts R. L., Mösch H. U., Fink G. R. 14-3-3 proteins are essential for RAS/MAPK cascade signaling during pseudohyphal development in S. cerevisiae. Cell. 1997 Jun 27;89(7):1055–1065. doi: 10.1016/s0092-8674(00)80293-7. [DOI] [PubMed] [Google Scholar]
  42. Rouse J., Jackson S. P. LCD1: an essential gene involved in checkpoint control and regulation of the MEC1 signalling pathway in Saccharomyces cerevisiae. EMBO J. 2000 Nov 1;19(21):5801–5812. doi: 10.1093/emboj/19.21.5801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Russell P. Checkpoints on the road to mitosis. Trends Biochem Sci. 1998 Oct;23(10):399–402. doi: 10.1016/s0968-0004(98)01291-2. [DOI] [PubMed] [Google Scholar]
  44. Sanchez Y., Bachant J., Wang H., Hu F., Liu D., Tetzlaff M., Elledge S. J. Control of the DNA damage checkpoint by chk1 and rad53 protein kinases through distinct mechanisms. Science. 1999 Nov 5;286(5442):1166–1171. doi: 10.1126/science.286.5442.1166. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. Santocanale C., Diffley J. F. A Mec1- and Rad53-dependent checkpoint controls late-firing origins of DNA replication. Nature. 1998 Oct 8;395(6702):615–618. doi: 10.1038/27001. [DOI] [PubMed] [Google Scholar]
  47. Santocanale C., Neecke H., Longhese M. P., Lucchini G., Plevani P. Mutations in the gene encoding the 34 kDa subunit of yeast replication protein A cause defective S phase progression. J Mol Biol. 1995 Dec 8;254(4):595–607. doi: 10.1006/jmbi.1995.0641. [DOI] [PubMed] [Google Scholar]
  48. 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]
  49. Struhl K., Davis R. W. A physical, genetic and transcriptional map of the cloned his3 gene region of Saccharomyces cerevisiae. J Mol Biol. 1980 Jan 25;136(3):309–332. doi: 10.1016/0022-2836(80)90376-9. [DOI] [PubMed] [Google Scholar]
  50. Sun Z., Hsiao J., Fay D. S., Stern D. F. Rad53 FHA domain associated with phosphorylated Rad9 in the DNA damage checkpoint. Science. 1998 Jul 10;281(5374):272–274. doi: 10.1126/science.281.5374.272. [DOI] [PubMed] [Google Scholar]
  51. Tzivion Guri, Avruch Joseph. 14-3-3 proteins: active cofactors in cellular regulation by serine/threonine phosphorylation. J Biol Chem. 2001 Nov 14;277(5):3061–3064. doi: 10.1074/jbc.R100059200. [DOI] [PubMed] [Google Scholar]
  52. Vialard J. E., Gilbert C. S., Green C. M., Lowndes N. F. The budding yeast Rad9 checkpoint protein is subjected to Mec1/Tel1-dependent hyperphosphorylation and interacts with Rad53 after DNA damage. EMBO J. 1998 Oct 1;17(19):5679–5688. doi: 10.1093/emboj/17.19.5679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Wach A., Brachat A., Pöhlmann R., Philippsen P. New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast. 1994 Dec;10(13):1793–1808. doi: 10.1002/yea.320101310. [DOI] [PubMed] [Google Scholar]
  54. Waga S., Stillman B. The DNA replication fork in eukaryotic cells. Annu Rev Biochem. 1998;67:721–751. doi: 10.1146/annurev.biochem.67.1.721. [DOI] [PubMed] [Google Scholar]
  55. Wakayama T., Kondo T., Ando S., Matsumoto K., Sugimoto K. Pie1, a protein interacting with Mec1, controls cell growth and checkpoint responses in Saccharomyces cerevisiae. Mol Cell Biol. 2001 Feb;21(3):755–764. doi: 10.1128/MCB.21.3.755-764.2001. [DOI] [PMC free article] [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. Yang J., Winkler K., Yoshida M., Kornbluth S. Maintenance of G2 arrest in the Xenopus oocyte: a role for 14-3-3-mediated inhibition of Cdc25 nuclear import. EMBO J. 1999 Apr 15;18(8):2174–2183. doi: 10.1093/emboj/18.8.2174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Zeng Y., Forbes K. C., Wu Z., Moreno S., Piwnica-Worms H., Enoch T. Replication checkpoint requires phosphorylation of the phosphatase Cdc25 by Cds1 or Chk1. Nature. 1998 Oct 1;395(6701):507–510. doi: 10.1038/26766. [DOI] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES