Current Genetics

, Volume 23, Issue 5–6, pp 402–407 | Cite as

The use of a double-marker shuttle vector to study DNA double-strand break repair in wild-type and radiation-sensitive mutants of the yeast Saccharomyces cerevisiae

  • Bhavanath Jha
  • Fred Ahne
  • Friederike Eckardt-Schupp
Original Articles

Abstract

An episomal DNA vector (YpJA18), encoding two selectable recombinant yeast genes (TRP1, URA3), was constructed to assess the fidelity of DNA repair in haploid repair-competent (RAD) wild-type yeast and several radiation-sensitive mutants. Either a DNA double-strand break (DSB) or a double-strand gap of 169 bp (DSG) was introduced by restriction enzymes in-vitro within the coding sequence of the URA3 gene of this vector. To eliminate transfer artefacts, selection was first applied for the undamaged TRP1 gene followed by counter selection for URA3 gene activity, which indicated correct repair of the DSB and DSG. Correct repair of the damaged URA3 gene was found to be about 90% in RAD cells (normalized for the expression of undamaged URA3 in TRP+ transformants). Plasmids isolated from the transformants (URA+TRP+) carry both unique sites (ApaI and NcoI) within the URA3 gene indicating the precise restitution of the 169-bp gap. An excision-repair-defective rad4-4 mutant repaired these lesions as correctly as RAD cells, whereas the mutants rad50-1, rad51-1 and rad54-1, proven to be defective in DSB repair and mitotic recombination, showed less than 5% correct repair of such lesions. In contrast, a representative of the RAD6 epistasis group of genes, the rev2-1 mutant which is sensitive towards UV and ionizing radiation, had a significantly reduced ability (about 20%) for the correct repair of both DSBs and DSGs.

Key words

Double-marker plasmid DNA double-strand break Yeast transformation Fidelity of repair 

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References

  1. Ahne F, Baur M, Eckardt-Schupp F (1992) Curr Genet 22:277–282Google Scholar
  2. Blöcher D, Pohlit W (1982) Int J Radiat Biol 42:329–338Google Scholar
  3. Bouffler SD, Jha B, Johnson RT (1990) Somatic Cell Mol Genet 16:451–460Google Scholar
  4. Boyer HW, Roulland-Dussoix D (1969) J Mol Biol 41:459–472Google Scholar
  5. Brendel M, Haynes RH (1973) Mol Gen Genet 125:197–216Google Scholar
  6. Budd M, Mortimer RK (1982) Mutat Res 103:19–29Google Scholar
  7. Cox R, Debenham PG, Masson WK, Webb MBT (1986) Mol Biol Med 3:229–244Google Scholar
  8. Debenham PG, Webb MBT, Jones NJ, Cox R (1987) J Cell Sci, suppl 6:177–189Google Scholar
  9. Debenham PG, Webb MBT, Strech A, Thacher J (1988) Mutat Res 199:145–158Google Scholar
  10. Eckardt-Schupp F, Ahne F (1993) Mutat Res (in press)Google Scholar
  11. Falco SC, Rose M, Botstein D (1983) Genetics 105:843–856Google Scholar
  12. Frankenberg D, Frankenberg-Schwager M, Blöcher D, Harbich R (1981) Radiat Res 88:524–532Google Scholar
  13. Frankenberg-Schwager M (1989) Radiother Oncol 14:307–320Google Scholar
  14. Friedberg EC (1988) Microbiol Rev 52:70–102Google Scholar
  15. Friedberg EC, Siede W, Cooper AJ (1991) In: Broach JR, Pringle JR, Jones EW (eds) The molecular and cell biology of the yeast Saccharomyces cerevisiae: genome, dynamics, protein synthesis, and energetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp 147–192Google Scholar
  16. Game JC (1983) In: Spencer JFT, Spencer DH, Smith ARW (eds) Yeast genetics — fundamental and applied aspects. Springer, Berlin Heidelberg New York, pp 109–137Google Scholar
  17. Game JC, Mortimer RK (1974) Mutat Res 24:281–292Google Scholar
  18. Geigl E-M, Eckardt-Schupp F (1990) Mol Microbiol 4 (5):801–810Google Scholar
  19. Geigl E-M, Eckardt-Schupp F (1991a) Mol Microbiol 5(7):1615–1620Google Scholar
  20. Geigl E-M, Eckardt-Schupp F (1991b) Curr Genet 20:33–37Google Scholar
  21. Glaser VM, Glasunov AV, Tevzadze GG, Perera JR, Shestakov SV (1990) Curr Genet 18:1–5Google Scholar
  22. Haynes R, Kunz B (1981) In: Strathern JN, Jones EW, Broach JR (eds) The molecular biology of the yeast Saccharomyces cerevisiae: life cycle and inheritance. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 371–414Google Scholar
  23. Hinnen A, Hicks JB, Fink GR (1978) Proc Natl Acad Sci USA 75:1929–1933Google Scholar
  24. Iliakis J, Blöcher D, Metzger L, Pantelias G (1991) Int J Radiat Biol 59:927–939Google Scholar
  25. Johnson RE, Henderson ST, Petes TD, Prakash S, Bankmann M, Prakash L (1992) Mol Cell Biol 12:3807–3818Google Scholar
  26. Kuo C, Campbell J (1983) Mol Cell Biol 3:1730–1737Google Scholar
  27. Lawrence C (1982) Adv Genet 21:173–254Google Scholar
  28. Lederberg E, Cohen S (1974) J Bacteriol 119:1072–1074Google Scholar
  29. Lemontt, JF (1971) Mutat Res 13:319–326Google Scholar
  30. Lemontt JF (1972) Mol Gen Genet 119:27–42Google Scholar
  31. Mc Kee RH, Lawrence CW (1980) Mutat Res 70:37–48Google Scholar
  32. Mezard C, Pompon D, Nicolas A (1992) Cell 70:659–670Google Scholar
  33. Orr-Weaver TL, Szostak JW (1983) Proc Natl Acad Sci USA 80:4417–4421Google Scholar
  34. Orr-Weaver TL, Szostak JW, Rothstein RJ (1981) Proc Natl Acad Sci USA 78:6354–6358Google Scholar
  35. Perera JR, Glasunov AV, Glaser VM, Boreiko AV (1988) Mol Gen Genet 213:421–424Google Scholar
  36. Petes TD, Malone RE, Lorraine SS (1991) In: Broach JR, Pringle JR, Jones EW (eds) The molecular and cell biology of the yeast Saccharomyces cerevisiae: genome dynamics, protein synthesis, and energetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp 407–521Google Scholar
  37. Resnick MA (1987) Investigating the genetic control of biochemical events in meiotic recombination. In: Moens PB (ed) Meiosis. Academic Press, New York, pp 157–210Google Scholar
  38. Resnick MA, Martin P (1976) Mol Gen Genet 143:119–129Google Scholar
  39. Resnick MA, Zgaga Z, Hieter P, Westmoreland J, Fogel S, Nilsson-Tillgren T (1992) Mol Gen Genet 234:65–73Google Scholar
  40. Rose M, Winston F (1984) Mol Gen Genet 193:557–560Google Scholar
  41. Rose M, Grisafi P, Botstein D (1984) Gene 29:113–124Google Scholar
  42. Saeki T, Machida I, Nakai S (1980) Mutat Res 73:251–265Google Scholar
  43. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  44. Siede W, Brendel M (1981) Curr Genet 4:145–149Google Scholar
  45. Siede W, Eckardt-Schupp F (1986) Curr Genet 11:205–210Google Scholar
  46. Thacker J (1989) Mutat Res 220:187–204Google Scholar
  47. Ward JF (1990) Int J Radiat Biol 57:1141–1150Google Scholar
  48. Wendel S (1990) PhD thesis, Ludwig-Maximilians-Universität, MünchenGoogle Scholar

Copyright information

© Springer-Verlag 1993

Authors and Affiliations

  • Bhavanath Jha
    • 1
  • Fred Ahne
    • 2
  • Friederike Eckardt-Schupp
    • 2
  1. 1.Botany DepartmentL. N. Mithila UniversityDarbhangaIndia
  2. 2.Institut für StrahlenbiologieGSF-Forschungszentrum für Umwelt- und GesundheitNeuherbergGermany

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