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.
Full text
PDF![1078](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e53c/365179/ca43d1e4e347/molcellb00075-0124.png)
![1079](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e53c/365179/dd9c8ebcf538/molcellb00075-0125.png)
![1080](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e53c/365179/2cffa907ec1a/molcellb00075-0126.png)
![1081](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e53c/365179/d23d1106b94c/molcellb00075-0127.png)
![1082](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e53c/365179/d6fbbdfd933c/molcellb00075-0128.png)
![1083](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e53c/365179/cbe401314da0/molcellb00075-0129.png)
![1084](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e53c/365179/d66497ffdc7a/molcellb00075-0130.png)
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- 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]
- BURNS V. W. X-ray-induced division delay of individual yeast cells. Radiat Res. 1956 May;4(5):394–412. [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]