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. 1987 Mar;7(3):1078–1084. doi: 10.1128/mcb.7.3.1078

Regulation of RAD54- and RAD52-lacZ gene fusions in Saccharomyces cerevisiae in response to DNA damage.

G M Cole, D Schild, S T Lovett, R K Mortimer
PMCID: PMC365179  PMID: 3550429

Abstract

The RAD52 and RAD54 genes in the yeast Saccharomyces cerevisiae are involved in both DNA repair and DNA recombination. RAD54 has recently been shown to be inducible by X-rays, while RAD52 is not. To further investigate the regulation of these genes, we constructed gene fusions using 5' regions upstream of the RAD52 and RAD54 genes and a 3'-terminal fragment of the Escherichia coli beta-galactosidase gene. Yeast transformants with either an integrated or an autonomously replicating plasmid containing these fusions expressed beta-galactosidase activity constitutively. In addition, the RAD54 gene fusion was inducible in both haploid and diploid cells in response to the DNA-damaging agents X-rays, UV light, and methyl methanesulfonate, but not in response to heat shock. The RAD52-lacZ gene fusion showed little or no induction in response to X-ray or UV radiation nor methyl methanesulfonate. Typical induction levels for RAD54 in cells exposed to such agents were from 3- to 12-fold, in good agreement with previous mRNA analyses. When MATa cells were arrested in G1 with alpha-factor, RAD54 was still inducible after DNA damage, indicating that the observed induction is independent of the cell cycle. Using a yeast vector containing the EcoRI structural gene fused to the GAL1 promoter, we showed that double-strand breaks alone are sufficient in vivo for induction of RAD54.

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Selected References

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

  1. Adzuma K., Ogawa T., Ogawa H. Primary structure of the RAD52 gene in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Dec;4(12):2735–2744. doi: 10.1128/mcb.4.12.2735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. BURNS V. W. X-ray-induced division delay of individual yeast cells. Radiat Res. 1956 May;4(5):394–412. [PubMed] [Google Scholar]
  3. Barnes G., Rine J. Regulated expression of endonuclease EcoRI in Saccharomyces cerevisiae: nuclear entry and biological consequences. Proc Natl Acad Sci U S A. 1985 Mar;82(5):1354–1358. doi: 10.1073/pnas.82.5.1354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brunborg G., Resnick M. A., Williamson D. H. Cell-cycle-specific repair of DNA double strand breaks in Saccharomyces cerevisiae. Radiat Res. 1980 Jun;82(3):547–558. [PubMed] [Google Scholar]
  5. Budd M., Mortimer R. K. The effect of cycloheximide on repair in a temperature conditional radiation-sensitive mutant of Saccharomyces cerevisiae. Radiat Res. 1984 Sep;99(3):582–590. [PubMed] [Google Scholar]
  6. Bücking-Throm E., Duntze W., Hartwell L. H., Manney T. R. Reversible arrest of haploid yeast cells in the initiation of DNA synthesis by a diffusible sex factor. Exp Cell Res. 1973 Jan;76(1):99–110. doi: 10.1016/0014-4827(73)90424-2. [DOI] [PubMed] [Google Scholar]
  7. Casadaban M. J., Martinez-Arias A., Shapira S. K., Chou J. Beta-galactosidase gene fusions for analyzing gene expression in escherichia coli and yeast. Methods Enzymol. 1983;100:293–308. doi: 10.1016/0076-6879(83)00063-4. [DOI] [PubMed] [Google Scholar]
  8. Game J. C., Cox B. S. Synergistic interactions between rad mutations in yeast. Mutat Res. 1973 Oct;20(1):35–44. doi: 10.1016/0027-5107(73)90095-x. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Game J. C., Zamb T. J., Braun R. J., Resnick M., Roth R. M. The Role of Radiation (rad) Genes in Meiotic Recombination in Yeast. Genetics. 1980 Jan;94(1):51–68. doi: 10.1093/genetics/94.1.51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Guarente L., Ptashne M. Fusion of Escherichia coli lacZ to the cytochrome c gene of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2199–2203. doi: 10.1073/pnas.78.4.2199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Guarente L. Yeast promoters and lacZ fusions designed to study expression of cloned genes in yeast. Methods Enzymol. 1983;101:181–191. doi: 10.1016/0076-6879(83)01013-7. [DOI] [PubMed] [Google Scholar]
  13. Guarente L. Yeast promoters: positive and negative elements. Cell. 1984 Apr;36(4):799–800. doi: 10.1016/0092-8674(84)90028-x. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. MORTIMER R. K. Radiobiological and genetic studies on a polyploid series (haploid to hexaploid) of Saccharomyces cerevisiae. Radiat Res. 1958 Sep;9(3):312–326. [PubMed] [Google Scholar]
  17. McClanahan T., McEntee K. Specific transcripts are elevated in Saccharomyces cerevisiae in response to DNA damage. Mol Cell Biol. 1984 Nov;4(11):2356–2363. doi: 10.1128/mcb.4.11.2356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Nakai S., Matsumoto S. Two types of radiation-sensitive mutant in yeast. Mutat Res. 1967 Mar-Apr;4(2):129–136. doi: 10.1016/0027-5107(67)90064-4. [DOI] [PubMed] [Google Scholar]
  19. Peterson T. A., Prakash L., Prakash S., Osley M. A., Reed S. I. Regulation of CDC9, the Saccharomyces cerevisiae gene that encodes DNA ligase. Mol Cell Biol. 1985 Jan;5(1):226–235. doi: 10.1128/mcb.5.1.226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Prakash S., Prakash L., Burke W., Montelone B. A. Effects of the RAD52 Gene on Recombination in SACCHAROMYCES CEREVISIAE. Genetics. 1980 Jan;94(1):31–50. doi: 10.1093/genetics/94.1.31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Robinson G. W., Nicolet C. M., Kalainov D., Friedberg E. C. A yeast excision-repair gene is inducible by DNA damaging agents. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1842–1846. doi: 10.1073/pnas.83.6.1842. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rose M., Casadaban M. J., Botstein D. Yeast genes fused to beta-galactosidase in Escherichia coli can be expressed normally in yeast. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2460–2464. doi: 10.1073/pnas.78.4.2460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ruby S. W., Szostak J. W., Murray A. W. Cloning regulated yeast genes from a pool of lacZ fusions. Methods Enzymol. 1983;101:253–269. doi: 10.1016/0076-6879(83)01019-8. [DOI] [PubMed] [Google Scholar]
  25. Ruby S. W., Szostak J. W. Specific Saccharomyces cerevisiae genes are expressed in response to DNA-damaging agents. Mol Cell Biol. 1985 Jan;5(1):75–84. doi: 10.1128/mcb.5.1.75. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sumrada R., Cooper T. G. Control of vacuole permeability and protein degradation by the cell cycle arrest signal in Saccharomyces cerevisiae. J Bacteriol. 1978 Oct;136(1):234–246. doi: 10.1128/jb.136.1.234-246.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Throm E., Duntze W. Mating-Type-Dependent Inhibition of Deoxyribonucleic Acid Synthesis in Saccharomyces cerevisiae. J Bacteriol. 1970 Dec;104(3):1388–1390. doi: 10.1128/jb.104.3.1388-1390.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Walker G. C. Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli. Microbiol Rev. 1984 Mar;48(1):60–93. doi: 10.1128/mr.48.1.60-93.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]

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