Skip to main content
Genetics logoLink to Genetics
. 1996 Jan;142(1):91–102. doi: 10.1093/genetics/142.1.91

The Saccharomyces Cerevisiae Ku Autoantigen Homologue Affects Radiosensitivity Only in the Absence of Homologous Recombination

W Siede 1, A A Friedl 1, I Dianova 1, F Eckardt-Schupp 1, E C Friedberg 1
PMCID: PMC1206967  PMID: 8770587

Abstract

In mammalian cells, all subunits of the DNA-dependent protein kinase (DNA-PK) have been implicated in the repair of DNA double-strand breaks and in V(D)J recombination. In the yeast Saccharomyces cerevisiae, we have examined the phenotype conferred by a deletion of HDF1, the putative homologue of the 70-kD subunit of the DNA-end binding Ku complex of DNA-PK. The yeast gene does not play a role in radiation-induced cell cycle checkpoint arrest in G(1) and G(2) or in hydroxyurea-induced checkpoint arrest in S. In cells competent for homologous recombination, we could not detect any sensitivity to ionizing radiation or to methyl methanesulfonate (MMS) conferred by a hdf1 deletion and indeed, the repair of DNA double-strand breaks was not impaired. However, if homologous recombination was disabled (rad52 mutant background), inactivation of HDF1 results in additional sensitization toward ionizing radiation and MMS. These results give further support to the notion that, in contrast to higher eukaryotic cells, homologous recombination is the favored pathway of double-strand break repair in yeast whereas other competing mechanisms such as the suggested pathway of DNA-PK-dependent direct break rejoining are only of minor importance.

Full Text

The Full Text of this article is available as a PDF (3.3 MB).

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. Almasan A., Linke S. P., Paulson T. G., Huang L. C., Wahl G. M. Genetic instability as a consequence of inappropriate entry into and progression through S-phase. Cancer Metastasis Rev. 1995 Mar;14(1):59–73. doi: 10.1007/BF00690212. [DOI] [PubMed] [Google Scholar]
  3. Anderson C. W., Lees-Miller S. P. The nuclear serine/threonine protein kinase DNA-PK. Crit Rev Eukaryot Gene Expr. 1992;2(4):283–314. [PubMed] [Google Scholar]
  4. Anderson C. W. Protein kinases and the response to DNA damage. Semin Cell Biol. 1994 Dec;5(6):427–436. doi: 10.1006/scel.1994.1050. [DOI] [PubMed] [Google Scholar]
  5. Bendixen C., Sunjevaric I., Bauchwitz R., Rothstein R. Identification of a mouse homologue of the Saccharomyces cerevisiae recombination and repair gene, RAD52. Genomics. 1994 Sep 1;23(1):300–303. doi: 10.1006/geno.1994.1503. [DOI] [PubMed] [Google Scholar]
  6. Bezzubova O. Y., Schmidt H., Ostermann K., Heyer W. D., Buerstedde J. M. Identification of a chicken RAD52 homologue suggests conservation of the RAD52 recombination pathway throughout the evolution of higher eukaryotes. Nucleic Acids Res. 1993 Dec 25;21(25):5945–5949. doi: 10.1093/nar/21.25.5945. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bezzubova O., Shinohara A., Mueller R. G., Ogawa H., Buerstedde J. M. A chicken RAD51 homologue is expressed at high levels in lymphoid and reproductive organs. Nucleic Acids Res. 1993 Apr 11;21(7):1577–1580. doi: 10.1093/nar/21.7.1577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Blunt T., Finnie N. J., Taccioli G. E., Smith G. C., Demengeot J., Gottlieb T. M., Mizuta R., Varghese A. J., Alt F. W., Jeggo P. A. Defective DNA-dependent protein kinase activity is linked to V(D)J recombination and DNA repair defects associated with the murine scid mutation. Cell. 1995 Mar 10;80(5):813–823. doi: 10.1016/0092-8674(95)90360-7. [DOI] [PubMed] [Google Scholar]
  9. Chlebowicz E., Jachymczyk W. J. Repair of MMS-induced DNA double-strand breaks in haploid cells of Saccharomyces cerevisiae, which requires the presence of a duplicate genome. Mol Gen Genet. 1979 Jan 2;167(3):279–286. doi: 10.1007/BF00267420. [DOI] [PubMed] [Google Scholar]
  10. Dardalhon M., Nohturfft A., Meniel V., Averbeck D. Repair of DNA double-strand breaks induced in Saccharomyces cerevisiae using different gamma-ray dose-rates: a pulsed-field gel electrophoresis analysis. Int J Radiat Biol. 1994 Mar;65(3):307–314. doi: 10.1080/09553009414550361. [DOI] [PubMed] [Google Scholar]
  11. Derbyshire M. K., Epstein L. H., Young C. S., Munz P. L., Fishel R. Nonhomologous recombination in human cells. Mol Cell Biol. 1994 Jan;14(1):156–169. doi: 10.1128/mcb.14.1.156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Esposito M. S., Ramirez R. M., Bruschi C. V. Recombinators, recombinases and recombination genes of yeasts. Curr Genet. 1994 Jan;25(1):1–11. doi: 10.1007/BF00712959. [DOI] [PubMed] [Google Scholar]
  13. Fairman M. P., Johnson A. P., Thacker J. Multiple components are involved in the efficient joining of double stranded DNA breaks in human cell extracts. Nucleic Acids Res. 1992 Aug 25;20(16):4145–4152. doi: 10.1093/nar/20.16.4145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Finnie N. J., Gottlieb T. M., Blunt T., Jeggo P. A., Jackson S. P. DNA-dependent protein kinase activity is absent in xrs-6 cells: implications for site-specific recombination and DNA double-strand break repair. Proc Natl Acad Sci U S A. 1995 Jan 3;92(1):320–324. doi: 10.1073/pnas.92.1.320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fishman-Lobell J., Haber J. E. Removal of nonhomologous DNA ends in double-strand break recombination: the role of the yeast ultraviolet repair gene RAD1. Science. 1992 Oct 16;258(5081):480–484. doi: 10.1126/science.1411547. [DOI] [PubMed] [Google Scholar]
  17. Fishman-Lobell J., Rudin N., Haber J. E. Two alternative pathways of double-strand break repair that are kinetically separable and independently modulated. Mol Cell Biol. 1992 Mar;12(3):1292–1303. doi: 10.1128/mcb.12.3.1292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Frankenberg-Schwager M., Frankenberg D., Blöcher D., Adamczyk C. Repair of DNA double-strand breaks in irradiated yeast cells under nongrowth conditions. Radiat Res. 1980 Jun;82(3):498–510. [PubMed] [Google Scholar]
  19. Frankenberg-Schwager M., Frankenberg D., Blöcher D., Adamczyk C. The influence of oxygen on the survival and yield of DNA double-strand breaks in irradiated yeast cells. Int J Radiat Biol Relat Stud Phys Chem Med. 1979 Sep;36(3):261–270. doi: 10.1080/09553007914551031. [DOI] [PubMed] [Google Scholar]
  20. Frankenberg-Schwager M., Frankenberg D. DNA double-strand breaks: their repair and relationship to cell killing in yeast. Int J Radiat Biol. 1990 Oct;58(4):569–575. doi: 10.1080/09553009014551931. [DOI] [PubMed] [Google Scholar]
  21. Frankenberg-Schwager M. Induction, repair and biological relevance of radiation-induced DNA lesions in eukaryotic cells. Radiat Environ Biophys. 1990;29(4):273–292. doi: 10.1007/BF01210408. [DOI] [PubMed] [Google Scholar]
  22. Game J. C. DNA double-strand breaks and the RAD50-RAD57 genes in Saccharomyces. Semin Cancer Biol. 1993 Apr;4(2):73–83. [PubMed] [Google Scholar]
  23. Ganesh A., North P., Thacker J. Repair and misrepair of site-specific DNA double-strand breaks by human cell extracts. Mutat Res. 1993 May;299(3-4):251–259. doi: 10.1016/0165-1218(93)90101-i. [DOI] [PubMed] [Google Scholar]
  24. Geigl E. M., Eckardt-Schupp F. The repair of double-strand breaks and S1 nuclease-sensitive sites can be monitored chromosome-specifically in Saccharomyces cerevisiae using pulse-field gel electrophoresis. Mol Microbiol. 1991 Jul;5(7):1615–1620. doi: 10.1111/j.1365-2958.1991.tb01908.x. [DOI] [PubMed] [Google Scholar]
  25. Getts R. C., Stamato T. D. Absence of a Ku-like DNA end binding activity in the xrs double-strand DNA repair-deficient mutant. J Biol Chem. 1994 Jun 10;269(23):15981–15984. [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Heyer W. D. The search for the right partner: homologous pairing and DNA strand exchange proteins in eukaryotes. Experientia. 1994 Mar 15;50(3):223–233. doi: 10.1007/BF01924005. [DOI] [PubMed] [Google Scholar]
  29. Ho K. S. Induction of DNA double-strand breaks by X-rays in a radiosensitive strain of the yeast Saccharomyces cerevisiae. Mutat Res. 1975 Dec;30(3):327–334. [PubMed] [Google Scholar]
  30. Humphrey T., Enoch T. Cell-cycle checkpoints. Keeping mitosis in check. Curr Biol. 1995 Apr 1;5(4):376–379. doi: 10.1016/s0960-9822(95)00077-7. [DOI] [PubMed] [Google Scholar]
  31. Jeggo P. A. X-ray sensitive mutants of Chinese hamster ovary cell line: radio-sensitivity of DNA synthesis. Mutat Res. 1985 May;145(3):171–176. doi: 10.1016/0167-8817(85)90024-0. [DOI] [PubMed] [Google Scholar]
  32. Kastan M. B., Zhan Q., el-Deiry W. S., Carrier F., Jacks T., Walsh W. V., Plunkett B. S., Vogelstein B., Fornace A. J., Jr A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell. 1992 Nov 13;71(4):587–597. doi: 10.1016/0092-8674(92)90593-2. [DOI] [PubMed] [Google Scholar]
  33. Kirchgessner C. U., Patil C. K., Evans J. W., Cuomo C. A., Fried L. M., Carter T., Oettinger M. A., Brown J. M. DNA-dependent kinase (p350) as a candidate gene for the murine SCID defect. Science. 1995 Feb 24;267(5201):1178–1183. doi: 10.1126/science.7855601. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. Lees-Miller S. P., Sakaguchi K., Ullrich S. J., Appella E., Anderson C. W. Human DNA-activated protein kinase phosphorylates serines 15 and 37 in the amino-terminal transactivation domain of human p53. Mol Cell Biol. 1992 Nov;12(11):5041–5049. doi: 10.1128/mcb.12.11.5041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Lu X., Lane D. P. Differential induction of transcriptionally active p53 following UV or ionizing radiation: defects in chromosome instability syndromes? Cell. 1993 Nov 19;75(4):765–778. doi: 10.1016/0092-8674(93)90496-d. [DOI] [PubMed] [Google Scholar]
  38. Malone R. E., Esposito R. E. The RAD52 gene is required for homothallic interconversion of mating types and spontaneous mitotic recombination in yeast. Proc Natl Acad Sci U S A. 1980 Jan;77(1):503–507. doi: 10.1073/pnas.77.1.503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Morita T., Yoshimura Y., Yamamoto A., Murata K., Mori M., Yamamoto H., Matsushiro A. A mouse homolog of the Escherichia coli recA and Saccharomyces cerevisiae RAD51 genes. Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6577–6580. doi: 10.1073/pnas.90.14.6577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Morris T., Thacker J. Formation of large deletions by illegitimate recombination in the HPRT gene of primary human fibroblasts. Proc Natl Acad Sci U S A. 1993 Feb 15;90(4):1392–1396. doi: 10.1073/pnas.90.4.1392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Mortimer R. K., Contopoulou R., Schild D. Mitotic chromosome loss in a radiation-sensitive strain of the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5778–5782. doi: 10.1073/pnas.78.9.5778. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Muris D. F., Bezzubova O., Buerstedde J. M., Vreeken K., Balajee A. S., Osgood C. J., Troelstra C., Hoeijmakers J. H., Ostermann K., Schmidt H. Cloning of human and mouse genes homologous to RAD52, a yeast gene involved in DNA repair and recombination. Mutat Res. 1994 Nov;315(3):295–305. doi: 10.1016/0921-8777(94)90040-x. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. Nicolás A. L., Munz P. L., Young C. S. A modified single-strand annealing model best explains the joining of DNA double-strand breaks mammalian cells and cell extracts. Nucleic Acids Res. 1995 Mar 25;23(6):1036–1043. doi: 10.1093/nar/23.6.1036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. North P., Ganesh A., Thacker J. The rejoining of double-strand breaks in DNA by human cell extracts. Nucleic Acids Res. 1990 Nov 11;18(21):6205–6210. doi: 10.1093/nar/18.21.6205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Ogawa T., Yu X., Shinohara A., Egelman E. H. Similarity of the yeast RAD51 filament to the bacterial RecA filament. Science. 1993 Mar 26;259(5103):1896–1899. doi: 10.1126/science.8456314. [DOI] [PubMed] [Google Scholar]
  47. Pergola F., Zdzienicka M. Z., Lieber M. R. V(D)J recombination in mammalian cell mutants defective in DNA double-strand break repair. Mol Cell Biol. 1993 Jun;13(6):3464–3471. doi: 10.1128/mcb.13.6.3464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Peterson S. R., Kurimasa A., Oshimura M., Dynan W. S., Bradbury E. M., Chen D. J. Loss of the catalytic subunit of the DNA-dependent protein kinase in DNA double-strand-break-repair mutant mammalian cells. Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3171–3174. doi: 10.1073/pnas.92.8.3171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Pfeiffer P., Vielmetter W. Joining of nonhomologous DNA double strand breaks in vitro. Nucleic Acids Res. 1988 Feb 11;16(3):907–924. doi: 10.1093/nar/16.3.907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Rathmell W. K., Chu G. Involvement of the Ku autoantigen in the cellular response to DNA double-strand breaks. Proc Natl Acad Sci U S A. 1994 Aug 2;91(16):7623–7627. doi: 10.1073/pnas.91.16.7623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. 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]
  52. Resnick M. A., Martin P. The repair of double-strand breaks in the nuclear DNA of Saccharomyces cerevisiae and its genetic control. Mol Gen Genet. 1976 Jan 16;143(2):119–129. doi: 10.1007/BF00266917. [DOI] [PubMed] [Google Scholar]
  53. Roth D. B., Lindahl T., Gellert M. Repair and recombination. How to make ends meet. Curr Biol. 1995 May 1;5(5):496–499. doi: 10.1016/s0960-9822(95)00101-1. [DOI] [PubMed] [Google Scholar]
  54. Roth D. B., Wilson J. H. Nonhomologous recombination in mammalian cells: role for short sequence homologies in the joining reaction. Mol Cell Biol. 1986 Dec;6(12):4295–4304. doi: 10.1128/mcb.6.12.4295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. 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]
  56. Shinohara A., Ogawa H., Ogawa T. Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein. Cell. 1992 May 1;69(3):457–470. doi: 10.1016/0092-8674(92)90447-k. [DOI] [PubMed] [Google Scholar]
  57. 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]
  58. 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]
  59. Siede W., Friedberg E. C. Regulation of the yeast RAD2 gene: DNA damage-dependent induction correlates with protein binding to regulatory sequences and their deletion influences survival. Mol Gen Genet. 1992 Mar;232(2):247–256. doi: 10.1007/BF00280003. [DOI] [PubMed] [Google Scholar]
  60. Smider V., Rathmell W. K., Lieber M. R., Chu G. Restoration of X-ray resistance and V(D)J recombination in mutant cells by Ku cDNA. Science. 1994 Oct 14;266(5183):288–291. doi: 10.1126/science.7939667. [DOI] [PubMed] [Google Scholar]
  61. Smith J., Rothstein R. A mutation in the gene encoding the Saccharomyces cerevisiae single-stranded DNA-binding protein Rfa1 stimulates a RAD52-independent pathway for direct-repeat recombination. Mol Cell Biol. 1995 Mar;15(3):1632–1641. doi: 10.1128/mcb.15.3.1632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Sugawara N., Ivanov E. L., Fishman-Lobell J., Ray B. L., Wu X., Haber J. E. DNA structure-dependent requirements for yeast RAD genes in gene conversion. Nature. 1995 Jan 5;373(6509):84–86. doi: 10.1038/373084a0. [DOI] [PubMed] [Google Scholar]
  63. Sung P. Catalysis of ATP-dependent homologous DNA pairing and strand exchange by yeast RAD51 protein. Science. 1994 Aug 26;265(5176):1241–1243. doi: 10.1126/science.8066464. [DOI] [PubMed] [Google Scholar]
  64. Suzuki K., Imai Y., Yamashita I., Fukui S. In vivo ligation of linear DNA molecules to circular forms in the yeast Saccharomyces cerevisiae. J Bacteriol. 1983 Aug;155(2):747–754. doi: 10.1128/jb.155.2.747-754.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Thompson L. H., Jeggo P. A. Nomenclature of human genes involved in ionizing radiation sensitivity. Mutat Res. 1995 Sep;337(2):131–134. doi: 10.1016/0921-8777(95)00018-f. [DOI] [PubMed] [Google Scholar]
  66. Tran H. T., Degtyareva N. P., Koloteva N. N., Sugino A., Masumoto H., Gordenin D. A., Resnick M. A. Replication slippage between distant short repeats in Saccharomyces cerevisiae depends on the direction of replication and the RAD50 and RAD52 genes. Mol Cell Biol. 1995 Oct;15(10):5607–5617. doi: 10.1128/mcb.15.10.5607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Ward J. F. DNA damage produced by ionizing radiation in mammalian cells: identities, mechanisms of formation, and reparability. Prog Nucleic Acid Res Mol Biol. 1988;35:95–125. doi: 10.1016/s0079-6603(08)60611-x. [DOI] [PubMed] [Google Scholar]
  68. Weiffenbach B., Haber J. E. Homothallic mating type switching generates lethal chromosome breaks in rad52 strains of Saccharomyces cerevisiae. Mol Cell Biol. 1981 Jun;1(6):522–534. doi: 10.1128/mcb.1.6.522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Weinert T., Lydall D. Cell cycle checkpoints, genetic instability and cancer. Semin Cancer Biol. 1993 Apr;4(2):129–140. [PubMed] [Google Scholar]
  70. Zdzienicka M. Z. Mammalian mutants defective in the response to ionizing radiation-induced DNA damage. Mutat Res. 1995 May;336(3):203–213. doi: 10.1016/0921-8777(95)00003-3. [DOI] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES