Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1998 Mar 16;17(6):1819–1828. doi: 10.1093/emboj/17.6.1819

Components of the Ku-dependent non-homologous end-joining pathway are involved in telomeric length maintenance and telomeric silencing.

S J Boulton 1, S P Jackson 1
PMCID: PMC1170529  PMID: 9501103

Abstract

In the budding yeast, Saccharomyces cerevisiae, genes in close proximity to telomeres are subject to transcriptional silencing through the process of telomere position effect (TPE). Here, we show that the protein Ku, previously implicated in DNA double-strand break (DSB) repair and in telomeric length maintenance, is also essential for telomeric silencing. Furthermore, using an in vivo plasmid rejoining assay, we demonstrate that SIR2, SIR3 and SIR4, three genes shown previously to function in TPE, are essential for Ku-dependent DSB repair. As is the case for Ku-deficient strains, residual repair operating in the absence of the SIR gene products ensues through an error-prone DNA repair pathway that results in terminal deletions. To identify novel components of the Ku-associated DSB repair pathway, we have tested several other candidate genes for their involvement in DNA DSB repair, telomeric maintenance and TPE. We show that TEL1, a gene required for telomeric length maintenance, is not required for either DNA DSB repair or TPE. However, RAD50, MRE11 and XRS2 function both in Ku-dependent DNA DSB repair and in telomeric length maintenance, although they have no major effects on TPE. These data provide important insights into DNA DSB repair and the linkage of this process to telomere length homeostasis and transcriptional silencing.

Full Text

The Full Text of this article is available as a PDF (332.7 KB).

Selected References

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

  1. Aparicio O. M., Billington B. L., Gottschling D. E. Modifiers of position effect are shared between telomeric and silent mating-type loci in S. cerevisiae. Cell. 1991 Sep 20;66(6):1279–1287. doi: 10.1016/0092-8674(91)90049-5. [DOI] [PubMed] [Google Scholar]
  2. Barnes G., Rio D. DNA double-strand-break sensitivity, DNA replication, and cell cycle arrest phenotypes of Ku-deficient Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1997 Feb 4;94(3):867–872. doi: 10.1073/pnas.94.3.867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blackburn E. H. Structure and function of telomeres. Nature. 1991 Apr 18;350(6319):569–573. doi: 10.1038/350569a0. [DOI] [PubMed] [Google Scholar]
  4. Boulton S. J., Jackson S. P. Identification of a Saccharomyces cerevisiae Ku80 homologue: roles in DNA double strand break rejoining and in telomeric maintenance. Nucleic Acids Res. 1996 Dec 1;24(23):4639–4648. doi: 10.1093/nar/24.23.4639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Boulton S. J., Jackson S. P. Saccharomyces cerevisiae Ku70 potentiates illegitimate DNA double-strand break repair and serves as a barrier to error-prone DNA repair pathways. EMBO J. 1996 Sep 16;15(18):5093–5103. [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Cohn M., Blackburn E. H. Telomerase in yeast. Science. 1995 Jul 21;269(5222):396–400. doi: 10.1126/science.7618104. [DOI] [PubMed] [Google Scholar]
  8. Connelly J. C., Leach D. R. The sbcC and sbcD genes of Escherichia coli encode a nuclease involved in palindrome inviability and genetic recombination. Genes Cells. 1996 Mar;1(3):285–291. doi: 10.1046/j.1365-2443.1996.23024.x. [DOI] [PubMed] [Google Scholar]
  9. Critchlow S. E., Bowater R. P., Jackson S. P. Mammalian DNA double-strand break repair protein XRCC4 interacts with DNA ligase IV. Curr Biol. 1997 Aug 1;7(8):588–598. doi: 10.1016/s0960-9822(06)00258-2. [DOI] [PubMed] [Google Scholar]
  10. Dolganov G. M., Maser R. S., Novikov A., Tosto L., Chong S., Bressan D. A., Petrini J. H. Human Rad50 is physically associated with human Mre11: identification of a conserved multiprotein complex implicated in recombinational DNA repair. Mol Cell Biol. 1996 Sep;16(9):4832–4841. doi: 10.1128/mcb.16.9.4832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dvir A., Stein L. Y., Calore B. L., Dynan W. S. Purification and characterization of a template-associated protein kinase that phosphorylates RNA polymerase II. J Biol Chem. 1993 May 15;268(14):10440–10447. [PubMed] [Google Scholar]
  12. Feldmann H., Driller L., Meier B., Mages G., Kellermann J., Winnacker E. L. HDF2, the second subunit of the Ku homologue from Saccharomyces cerevisiae. J Biol Chem. 1996 Nov 1;271(44):27765–27769. doi: 10.1074/jbc.271.44.27765. [DOI] [PubMed] [Google Scholar]
  13. Feldmann H., Winnacker E. L. A putative homologue of the human autoantigen Ku from Saccharomyces cerevisiae. J Biol Chem. 1993 Jun 15;268(17):12895–12900. [PubMed] [Google Scholar]
  14. Goffeau A., Barrell B. G., Bussey H., Davis R. W., Dujon B., Feldmann H., Galibert F., Hoheisel J. D., Jacq C., Johnston M. Life with 6000 genes. Science. 1996 Oct 25;274(5287):546, 563-7. doi: 10.1126/science.274.5287.546. [DOI] [PubMed] [Google Scholar]
  15. Gottlieb T. M., Jackson S. P. The DNA-dependent protein kinase: requirement for DNA ends and association with Ku antigen. Cell. 1993 Jan 15;72(1):131–142. doi: 10.1016/0092-8674(93)90057-w. [DOI] [PubMed] [Google Scholar]
  16. Gottschling D. E., Aparicio O. M., Billington B. L., Zakian V. A. Position effect at S. cerevisiae telomeres: reversible repression of Pol II transcription. Cell. 1990 Nov 16;63(4):751–762. doi: 10.1016/0092-8674(90)90141-z. [DOI] [PubMed] [Google Scholar]
  17. Grawunder U., Wilm M., Wu X., Kulesza P., Wilson T. E., Mann M., Lieber M. R. Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells. Nature. 1997 Jul 31;388(6641):492–495. doi: 10.1038/41358. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Grunstein M. Histone acetylation in chromatin structure and transcription. Nature. 1997 Sep 25;389(6649):349–352. doi: 10.1038/38664. [DOI] [PubMed] [Google Scholar]
  20. Hartley K. O., Gell D., Smith G. C., Zhang H., Divecha N., Connelly M. A., Admon A., Lees-Miller S. P., Anderson C. W., Jackson S. P. DNA-dependent protein kinase catalytic subunit: a relative of phosphatidylinositol 3-kinase and the ataxia telangiectasia gene product. Cell. 1995 Sep 8;82(5):849–856. doi: 10.1016/0092-8674(95)90482-4. [DOI] [PubMed] [Google Scholar]
  21. Hecht A., Laroche T., Strahl-Bolsinger S., Gasser S. M., Grunstein M. Histone H3 and H4 N-termini interact with SIR3 and SIR4 proteins: a molecular model for the formation of heterochromatin in yeast. Cell. 1995 Feb 24;80(4):583–592. doi: 10.1016/0092-8674(95)90512-x. [DOI] [PubMed] [Google Scholar]
  22. Jackson S. P. DNA damage detection by DNA dependent protein kinase and related enzymes. Cancer Surv. 1996;28:261–279. [PubMed] [Google Scholar]
  23. Jackson S. P. Genomic stability. Silencing and DNA repair connect. Nature. 1997 Aug 28;388(6645):829–830. doi: 10.1038/42136. [DOI] [PubMed] [Google Scholar]
  24. Jackson S. P., Jeggo P. A. DNA double-strand break repair and V(D)J recombination: involvement of DNA-PK. Trends Biochem Sci. 1995 Oct;20(10):412–415. doi: 10.1016/s0968-0004(00)89090-8. [DOI] [PubMed] [Google Scholar]
  25. Klar A. J., Fogel S., Macleod K. MAR1-a Regulator of the HMa and HMalpha Loci in SACCHAROMYCES CEREVISIAE. Genetics. 1979 Sep;93(1):37–50. doi: 10.1093/genetics/93.1.37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kramer K. M., Brock J. A., Bloom K., Moore J. K., Haber J. E. Two different types of double-strand breaks in Saccharomyces cerevisiae are repaired by similar RAD52-independent, nonhomologous recombination events. Mol Cell Biol. 1994 Feb;14(2):1293–1301. doi: 10.1128/mcb.14.2.1293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kuhn A., Gottlieb T. M., Jackson S. P., Grummt I. DNA-dependent protein kinase: a potent inhibitor of transcription by RNA polymerase I. Genes Dev. 1995 Jan 15;9(2):193–203. doi: 10.1101/gad.9.2.193. [DOI] [PubMed] [Google Scholar]
  28. Labhart P. DNA-dependent protein kinase specifically represses promoter-directed transcription initiation by RNA polymerase I. Proc Natl Acad Sci U S A. 1995 Mar 28;92(7):2934–2938. doi: 10.1073/pnas.92.7.2934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lieber M. R., Grawunder U., Wu X., Yaneva M. Tying loose ends: roles of Ku and DNA-dependent protein kinase in the repair of double-strand breaks. Curr Opin Genet Dev. 1997 Feb;7(1):99–104. doi: 10.1016/s0959-437x(97)80116-5. [DOI] [PubMed] [Google Scholar]
  30. Mages G. J., Feldmann H. M., Winnacker E. L. Involvement of the Saccharomyces cerevisiae HDF1 gene in DNA double-strand break repair and recombination. J Biol Chem. 1996 Apr 5;271(14):7910–7915. doi: 10.1074/jbc.271.14.7910. [DOI] [PubMed] [Google Scholar]
  31. Marcand S., Gilson E., Shore D. A protein-counting mechanism for telomere length regulation in yeast. Science. 1997 Feb 14;275(5302):986–990. doi: 10.1126/science.275.5302.986. [DOI] [PubMed] [Google Scholar]
  32. Mezard C., Nicolas A. Homologous, homeologous, and illegitimate repair of double-strand breaks during transformation of a wild-type strain and a rad52 mutant strain of Saccharomyces cerevisiae. Mol Cell Biol. 1994 Feb;14(2):1278–1292. doi: 10.1128/mcb.14.2.1278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Milne G. T., Jin S., Shannon K. B., Weaver D. T. Mutations in two Ku homologs define a DNA end-joining repair pathway in Saccharomyces cerevisiae. Mol Cell Biol. 1996 Aug;16(8):4189–4198. doi: 10.1128/mcb.16.8.4189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Moore J. K., Haber J. E. Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae. Mol Cell Biol. 1996 May;16(5):2164–2173. doi: 10.1128/mcb.16.5.2164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Moretti P., Freeman K., Coodly L., Shore D. Evidence that a complex of SIR proteins interacts with the silencer and telomere-binding protein RAP1. Genes Dev. 1994 Oct 1;8(19):2257–2269. doi: 10.1101/gad.8.19.2257. [DOI] [PubMed] [Google Scholar]
  36. Petrini J. H., Walsh M. E., DiMare C., Chen X. N., Korenberg J. R., Weaver D. T. Isolation and characterization of the human MRE11 homologue. Genomics. 1995 Sep 1;29(1):80–86. doi: 10.1006/geno.1995.1217. [DOI] [PubMed] [Google Scholar]
  37. Porter S. E., Greenwell P. W., Ritchie K. B., Petes T. D. The DNA-binding protein Hdf1p (a putative Ku homologue) is required for maintaining normal telomere length in Saccharomyces cerevisiae. Nucleic Acids Res. 1996 Feb 15;24(4):582–585. doi: 10.1093/nar/24.4.582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Rine J., Strathern J. N., Hicks J. B., Herskowitz I. A suppressor of mating-type locus mutations in Saccharomyces cerevisiae: evidence for and identification of cryptic mating-type loci. Genetics. 1979 Dec;93(4):877–901. doi: 10.1093/genetics/93.4.877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Schiestl R. H., Dominska M., Petes T. D. Transformation of Saccharomyces cerevisiae with nonhomologous DNA: illegitimate integration of transforming DNA into yeast chromosomes and in vivo ligation of transforming DNA to mitochondrial DNA sequences. Mol Cell Biol. 1993 May;13(5):2697–2705. doi: 10.1128/mcb.13.5.2697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Schiestl R. H., Petes T. D. Integration of DNA fragments by illegitimate recombination in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7585–7589. doi: 10.1073/pnas.88.17.7585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schiestl R. H., Zhu J., Petes T. D. Effect of mutations in genes affecting homologous recombination on restriction enzyme-mediated and illegitimate recombination in Saccharomyces cerevisiae. Mol Cell Biol. 1994 Jul;14(7):4493–4500. doi: 10.1128/mcb.14.7.4493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Schär P., Herrmann G., Daly G., Lindahl T. A newly identified DNA ligase of Saccharomyces cerevisiae involved in RAD52-independent repair of DNA double-strand breaks. Genes Dev. 1997 Aug 1;11(15):1912–1924. doi: 10.1101/gad.11.15.1912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Shinohara A., Ogawa T. Homologous recombination and the roles of double-strand breaks. Trends Biochem Sci. 1995 Oct;20(10):387–391. doi: 10.1016/s0968-0004(00)89085-4. [DOI] [PubMed] [Google Scholar]
  44. Shore D., Nasmyth K. Purification and cloning of a DNA binding protein from yeast that binds to both silencer and activator elements. Cell. 1987 Dec 4;51(5):721–732. doi: 10.1016/0092-8674(87)90095-x. [DOI] [PubMed] [Google Scholar]
  45. Shore D., Squire M., Nasmyth K. A. Characterization of two genes required for the position-effect control of yeast mating-type genes. EMBO J. 1984 Dec 1;3(12):2817–2823. doi: 10.1002/j.1460-2075.1984.tb02214.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Siede W., Friedl A. A., Dianova I., Eckardt-Schupp F., Friedberg E. C. The Saccharomyces cerevisiae Ku autoantigen homologue affects radiosensitivity only in the absence of homologous recombination. Genetics. 1996 Jan;142(1):91–102. doi: 10.1093/genetics/142.1.91. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Stavenhagen J. B., Zakian V. A. Internal tracts of telomeric DNA act as silencers in Saccharomyces cerevisiae. Genes Dev. 1994 Jun 15;8(12):1411–1422. doi: 10.1101/gad.8.12.1411. [DOI] [PubMed] [Google Scholar]
  48. Sun Z., Fay D. S., Marini F., Foiani M., Stern D. F. Spk1/Rad53 is regulated by Mec1-dependent protein phosphorylation in DNA replication and damage checkpoint pathways. Genes Dev. 1996 Feb 15;10(4):395–406. doi: 10.1101/gad.10.4.395. [DOI] [PubMed] [Google Scholar]
  49. Teo S. H., Jackson S. P. Identification of Saccharomyces cerevisiae DNA ligase IV: involvement in DNA double-strand break repair. EMBO J. 1997 Aug 1;16(15):4788–4795. doi: 10.1093/emboj/16.15.4788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Tsukamoto Y., Kato J., Ikeda H. Silencing factors participate in DNA repair and recombination in Saccharomyces cerevisiae. Nature. 1997 Aug 28;388(6645):900–903. doi: 10.1038/42288. [DOI] [PubMed] [Google Scholar]
  51. Wiley E. A., Zakian V. A. Extra telomeres, but not internal tracts of telomeric DNA, reduce transcriptional repression at Saccharomyces telomeres. Genetics. 1995 Jan;139(1):67–79. doi: 10.1093/genetics/139.1.67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wilson T. E., Grawunder U., Lieber M. R. Yeast DNA ligase IV mediates non-homologous DNA end joining. Nature. 1997 Jul 31;388(6641):495–498. doi: 10.1038/41365. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES