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.
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References
Ahne F, Baur M, Eckardt-Schupp F (1992) Curr Genet 22:277–282
Blöcher D, Pohlit W (1982) Int J Radiat Biol 42:329–338
Bouffler SD, Jha B, Johnson RT (1990) Somatic Cell Mol Genet 16:451–460
Boyer HW, Roulland-Dussoix D (1969) J Mol Biol 41:459–472
Brendel M, Haynes RH (1973) Mol Gen Genet 125:197–216
Budd M, Mortimer RK (1982) Mutat Res 103:19–29
Cox R, Debenham PG, Masson WK, Webb MBT (1986) Mol Biol Med 3:229–244
Debenham PG, Webb MBT, Jones NJ, Cox R (1987) J Cell Sci, suppl 6:177–189
Debenham PG, Webb MBT, Strech A, Thacher J (1988) Mutat Res 199:145–158
Eckardt-Schupp F, Ahne F (1993) Mutat Res (in press)
Falco SC, Rose M, Botstein D (1983) Genetics 105:843–856
Frankenberg D, Frankenberg-Schwager M, Blöcher D, Harbich R (1981) Radiat Res 88:524–532
Frankenberg-Schwager M (1989) Radiother Oncol 14:307–320
Friedberg EC (1988) Microbiol Rev 52:70–102
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 147–192
Game JC (1983) In: Spencer JFT, Spencer DH, Smith ARW (eds) Yeast genetics — fundamental and applied aspects. Springer, Berlin Heidelberg New York, pp 109–137
Game JC, Mortimer RK (1974) Mutat Res 24:281–292
Geigl E-M, Eckardt-Schupp F (1990) Mol Microbiol 4 (5):801–810
Geigl E-M, Eckardt-Schupp F (1991a) Mol Microbiol 5(7):1615–1620
Geigl E-M, Eckardt-Schupp F (1991b) Curr Genet 20:33–37
Glaser VM, Glasunov AV, Tevzadze GG, Perera JR, Shestakov SV (1990) Curr Genet 18:1–5
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, 371–414
Hinnen A, Hicks JB, Fink GR (1978) Proc Natl Acad Sci USA 75:1929–1933
Iliakis J, Blöcher D, Metzger L, Pantelias G (1991) Int J Radiat Biol 59:927–939
Johnson RE, Henderson ST, Petes TD, Prakash S, Bankmann M, Prakash L (1992) Mol Cell Biol 12:3807–3818
Kuo C, Campbell J (1983) Mol Cell Biol 3:1730–1737
Lawrence C (1982) Adv Genet 21:173–254
Lederberg E, Cohen S (1974) J Bacteriol 119:1072–1074
Lemontt, JF (1971) Mutat Res 13:319–326
Lemontt JF (1972) Mol Gen Genet 119:27–42
Mc Kee RH, Lawrence CW (1980) Mutat Res 70:37–48
Mezard C, Pompon D, Nicolas A (1992) Cell 70:659–670
Orr-Weaver TL, Szostak JW (1983) Proc Natl Acad Sci USA 80:4417–4421
Orr-Weaver TL, Szostak JW, Rothstein RJ (1981) Proc Natl Acad Sci USA 78:6354–6358
Perera JR, Glasunov AV, Glaser VM, Boreiko AV (1988) Mol Gen Genet 213:421–424
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 407–521
Resnick MA (1987) Investigating the genetic control of biochemical events in meiotic recombination. In: Moens PB (ed) Meiosis. Academic Press, New York, pp 157–210
Resnick MA, Martin P (1976) Mol Gen Genet 143:119–129
Resnick MA, Zgaga Z, Hieter P, Westmoreland J, Fogel S, Nilsson-Tillgren T (1992) Mol Gen Genet 234:65–73
Rose M, Winston F (1984) Mol Gen Genet 193:557–560
Rose M, Grisafi P, Botstein D (1984) Gene 29:113–124
Saeki T, Machida I, Nakai S (1980) Mutat Res 73:251–265
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Siede W, Brendel M (1981) Curr Genet 4:145–149
Siede W, Eckardt-Schupp F (1986) Curr Genet 11:205–210
Thacker J (1989) Mutat Res 220:187–204
Ward JF (1990) Int J Radiat Biol 57:1141–1150
Wendel S (1990) PhD thesis, Ludwig-Maximilians-Universität, München
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Jha, B., Ahne, F. & Eckardt-Schupp, F. 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 . Curr Genet 23, 402–407 (1993). https://doi.org/10.1007/BF00312626
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DOI: https://doi.org/10.1007/BF00312626