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. 2002 Oct;162(2):677–688. doi: 10.1093/genetics/162.2.677

A genomics-based screen for yeast mutants with an altered recombination/end-joining repair ratio.

Thomas E Wilson 1
PMCID: PMC1462309  PMID: 12399380

Abstract

We recently described a yeast assay suitable for genetic screening in which simple religation nonhomologous end-joining (NHEJ) and single-strand annealing (SSA) compete for repair of an I-SceI-created double-strand break. Here, the required allele has been introduced into an array of 4781 MATa deletion mutants and each strain screened individually. Two mutants (rad52 and srs2) showed a clear increase in the NHEJ/SSA ratio due to preferential impairment of SSA, but no mutant increased the absolute frequency of NHEJ significantly above the wild-type level. Seven mutants showed a decreased NHEJ/SSA ratio due to frank loss of NHEJ, which corresponded to all known structural/catalytic NHEJ components (yku70, yku80, dnl4, lif1, rad50, mre11, and xrs2); no new mutants in this category were identified. A clearly separable and surprisingly large set of 16 other mutants showed partial defects in NHEJ. Further examination of these revealed that NEJ1 can entirely account for the mating-type regulation of NHEJ, but that this regulatory role was distinct from the postdiauxic/stationary-phase induction of NHEJ that was deficient in other mutants (especially doa1, fyv6, and mck1). These results are discussed in the context of the minimal set of required proteins and regulatory inputs for NHEJ.

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Selected References

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  1. Ahne F., Jha B., Eckardt-Schupp F. The RAD5 gene product is involved in the avoidance of non-homologous end-joining of DNA double strand breaks in the yeast Saccharomyces cerevisiae. Nucleic Acids Res. 1997 Feb 15;25(4):743–749. doi: 10.1093/nar/25.4.743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bennett C. B., Lewis L. K., Karthikeyan G., Lobachev K. S., Jin Y. H., Sterling J. F., Snipe J. R., Resnick M. A. Genes required for ionizing radiation resistance in yeast. Nat Genet. 2001 Dec;29(4):426–434. doi: 10.1038/ng778. [DOI] [PubMed] [Google Scholar]
  3. Brachmann C. B., Davies A., Cost G. J., Caputo E., Li J., Hieter P., Boeke J. D. Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast. 1998 Jan 30;14(2):115–132. doi: 10.1002/(SICI)1097-0061(19980130)14:2<115::AID-YEA204>3.0.CO;2-2. [DOI] [PubMed] [Google Scholar]
  4. Chen L., Trujillo K., Ramos W., Sung P., Tomkinson A. E. Promotion of Dnl4-catalyzed DNA end-joining by the Rad50/Mre11/Xrs2 and Hdf1/Hdf2 complexes. Mol Cell. 2001 Nov;8(5):1105–1115. doi: 10.1016/s1097-2765(01)00388-4. [DOI] [PubMed] [Google Scholar]
  5. Davis A. P., Symington L. S. The yeast recombinational repair protein Rad59 interacts with Rad52 and stimulates single-strand annealing. Genetics. 2001 Oct;159(2):515–525. doi: 10.1093/genetics/159.2.515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Frank-Vaillant M., Marcand S. NHEJ regulation by mating type is exercised through a novel protein, Lif2p, essential to the ligase IV pathway. Genes Dev. 2001 Nov 15;15(22):3005–3012. doi: 10.1101/gad.206801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Game J. C., Mortimer R. K. A genetic study of x-ray sensitive mutants in yeast. Mutat Res. 1974 Sep;24(3):281–292. doi: 10.1016/0027-5107(74)90176-6. [DOI] [PubMed] [Google Scholar]
  8. Ghislain M., Dohmen R. J., Levy F., Varshavsky A. Cdc48p interacts with Ufd3p, a WD repeat protein required for ubiquitin-mediated proteolysis in Saccharomyces cerevisiae. EMBO J. 1996 Sep 16;15(18):4884–4899. [PMC free article] [PubMed] [Google Scholar]
  9. Görlich D., Kutay U. Transport between the cell nucleus and the cytoplasm. Annu Rev Cell Dev Biol. 1999;15:607–660. doi: 10.1146/annurev.cellbio.15.1.607. [DOI] [PubMed] [Google Scholar]
  10. Hegde V., Klein H. Requirement for the SRS2 DNA helicase gene in non-homologous end joining in yeast. Nucleic Acids Res. 2000 Jul 15;28(14):2779–2783. doi: 10.1093/nar/28.14.2779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Herrmann G., Lindahl T., Schär P. Saccharomyces cerevisiae LIF1: a function involved in DNA double-strand break repair related to mammalian XRCC4. EMBO J. 1998 Jul 15;17(14):4188–4198. doi: 10.1093/emboj/17.14.4188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Huang Juren, Dynan William S. Reconstitution of the mammalian DNA double-strand break end-joining reaction reveals a requirement for an Mre11/Rad50/NBS1-containing fraction. Nucleic Acids Res. 2002 Feb 1;30(3):667–674. doi: 10.1093/nar/30.3.667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ivanov E. L., Sugawara N., Fishman-Lobell J., Haber J. E. Genetic requirements for the single-strand annealing pathway of double-strand break repair in Saccharomyces cerevisiae. Genetics. 1996 Mar;142(3):693–704. doi: 10.1093/genetics/142.3.693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jackson S. P. Detecting, signalling and repairing DNA double-strand breaks. Biochem Soc Trans. 2001 Nov;29(Pt 6):655–661. doi: 10.1042/0300-5127:0290655. [DOI] [PubMed] [Google Scholar]
  15. Johnson E. S., Ma P. C., Ota I. M., Varshavsky A. A proteolytic pathway that recognizes ubiquitin as a degradation signal. J Biol Chem. 1995 Jul 21;270(29):17442–17456. doi: 10.1074/jbc.270.29.17442. [DOI] [PubMed] [Google Scholar]
  16. Jones J. M., Gellert M., Yang W. A Ku bridge over broken DNA. Structure. 2001 Oct;9(10):881–884. doi: 10.1016/s0969-2126(01)00658-x. [DOI] [PubMed] [Google Scholar]
  17. Karathanasis Elissa, Wilson Thomas E. Enhancement of Saccharomyces cerevisiae end-joining efficiency by cell growth stage but not by impairment of recombination. Genetics. 2002 Jul;161(3):1015–1027. doi: 10.1093/genetics/161.3.1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kegel A., Sjöstrand J. O., Aström S. U. Nej1p, a cell type-specific regulator of nonhomologous end joining in yeast. Curr Biol. 2001 Oct 16;11(20):1611–1617. doi: 10.1016/s0960-9822(01)00488-2. [DOI] [PubMed] [Google Scholar]
  19. Lee S. E., Pâques F., Sylvan J., Haber J. E. Role of yeast SIR genes and mating type in directing DNA double-strand breaks to homologous and non-homologous repair paths. Curr Biol. 1999 Jul 15;9(14):767–770. doi: 10.1016/s0960-9822(99)80339-x. [DOI] [PubMed] [Google Scholar]
  20. Lewis L. Kevin, Karthikeyan G., Westmoreland James W., Resnick Michael A. Differential suppression of DNA repair deficiencies of Yeast rad50, mre11 and xrs2 mutants by EXO1 and TLC1 (the RNA component of telomerase). Genetics. 2002 Jan;160(1):49–62. doi: 10.1093/genetics/160.1.49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ma Yunmei, Pannicke Ulrich, Schwarz Klaus, Lieber Michael R. Hairpin opening and overhang processing by an Artemis/DNA-dependent protein kinase complex in nonhomologous end joining and V(D)J recombination. Cell. 2002 Mar 22;108(6):781–794. doi: 10.1016/s0092-8674(02)00671-2. [DOI] [PubMed] [Google Scholar]
  22. Mahajan Kiran N., Nick McElhinny Stephanie A., Mitchell Beverly S., Ramsden Dale A. Association of DNA polymerase mu (pol mu) with Ku and ligase IV: role for pol mu in end-joining double-strand break repair. Mol Cell Biol. 2002 Jul;22(14):5194–5202. doi: 10.1128/MCB.22.14.5194-5202.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ni L., Snyder M. A genomic study of the bipolar bud site selection pattern in Saccharomyces cerevisiae. Mol Biol Cell. 2001 Jul;12(7):2147–2170. doi: 10.1091/mbc.12.7.2147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ooi S. L., Shoemaker D. D., Boeke J. D. A DNA microarray-based genetic screen for nonhomologous end-joining mutants in Saccharomyces cerevisiae. Science. 2001 Nov 8;294(5551):2552–2556. doi: 10.1126/science.1065672. [DOI] [PubMed] [Google Scholar]
  25. Ozkaynak E., Finley D., Solomon M. J., Varshavsky A. The yeast ubiquitin genes: a family of natural gene fusions. EMBO J. 1987 May;6(5):1429–1439. doi: 10.1002/j.1460-2075.1987.tb02384.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Peck V. M., Fuge E. K., Padilla P. A., Gomez M. A., Werner-Washburne M. Yeast bcy1 mutants with stationary phase-specific defects. Curr Genet. 1997 Aug;32(2):83–92. doi: 10.1007/s002940050251. [DOI] [PubMed] [Google Scholar]
  27. Planta R. J., Brown A. J., Cadahia J. L., Cerdan M. E., de Jonge M., Gent M. E., Hayes A., Kolen C. P., Lombardia L. J., Sefton M. Transcript analysis of 250 novel yeast genes from chromosome XIV. Yeast. 1999 Mar 15;15(4):329–350. doi: 10.1002/(SICI)1097-0061(19990315)15:4<329::AID-YEA360>3.0.CO;2-C. [DOI] [PubMed] [Google Scholar]
  28. Pâques F., Haber J. E. Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol Mol Biol Rev. 1999 Jun;63(2):349–404. doi: 10.1128/mmbr.63.2.349-404.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Shinohara A., Shinohara M., Ohta T., Matsuda S., Ogawa T. Rad52 forms ring structures and co-operates with RPA in single-strand DNA annealing. Genes Cells. 1998 Mar;3(3):145–156. doi: 10.1046/j.1365-2443.1998.00176.x. [DOI] [PubMed] [Google Scholar]
  30. Smith G. C., Jackson S. P. The DNA-dependent protein kinase. Genes Dev. 1999 Apr 15;13(8):916–934. doi: 10.1101/gad.13.8.916. [DOI] [PubMed] [Google Scholar]
  31. Smith J., Rothstein R. An allele of RFA1 suppresses RAD52-dependent double-strand break repair in Saccharomyces cerevisiae. Genetics. 1999 Feb;151(2):447–458. doi: 10.1093/genetics/151.2.447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sugawara N., Ira G., Haber J. E. DNA length dependence of the single-strand annealing pathway and the role of Saccharomyces cerevisiae RAD59 in double-strand break repair. Mol Cell Biol. 2000 Jul;20(14):5300–5309. doi: 10.1128/mcb.20.14.5300-5309.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Thompson L. H., Rubin J. S., Cleaver J. E., Whitmore G. F., Brookman K. A screening method for isolating DNA repair-deficient mutants of CHO cells. Somatic Cell Genet. 1980 May;6(3):391–405. doi: 10.1007/BF01542791. [DOI] [PubMed] [Google Scholar]
  34. Tong A. H., Evangelista M., Parsons A. B., Xu H., Bader G. D., Pagé N., Robinson M., Raghibizadeh S., Hogue C. W., Bussey H. Systematic genetic analysis with ordered arrays of yeast deletion mutants. Science. 2001 Dec 14;294(5550):2364–2368. doi: 10.1126/science.1065810. [DOI] [PubMed] [Google Scholar]
  35. Uetz P., Giot L., Cagney G., Mansfield T. A., Judson R. S., Knight J. R., Lockshon D., Narayan V., Srinivasan M., Pochart P. A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature. 2000 Feb 10;403(6770):623–627. doi: 10.1038/35001009. [DOI] [PubMed] [Google Scholar]
  36. Valencia M., Bentele M., Vaze M. B., Herrmann G., Kraus E., Lee S. E., Schär P., Haber J. E. NEJ1 controls non-homologous end joining in Saccharomyces cerevisiae. Nature. 2001 Dec 6;414(6864):666–669. doi: 10.1038/414666a. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Wilson T. E., Lieber M. R. Efficient processing of DNA ends during yeast nonhomologous end joining. Evidence for a DNA polymerase beta (Pol4)-dependent pathway. J Biol Chem. 1999 Aug 13;274(33):23599–23609. doi: 10.1074/jbc.274.33.23599. [DOI] [PubMed] [Google Scholar]
  39. Winzeler E. A., Shoemaker D. D., Astromoff A., Liang H., Anderson K., Andre B., Bangham R., Benito R., Boeke J. D., Bussey H. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science. 1999 Aug 6;285(5429):901–906. doi: 10.1126/science.285.5429.901. [DOI] [PubMed] [Google Scholar]
  40. de la Torre-Ruiz M., Lowndes N. F. The Saccharomyces cerevisiae DNA damage checkpoint is required for efficient repair of double strand breaks by non-homologous end joining. FEBS Lett. 2000 Feb 11;467(2-3):311–315. doi: 10.1016/s0014-5793(00)01180-7. [DOI] [PubMed] [Google Scholar]

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