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. 1996 May;16(5):2091–2100. doi: 10.1128/mcb.16.5.2091

The Saccharomyces cerevisiae IMP2 gene encodes a transcriptional activator that mediates protection against DNA damage caused by bleomycin and other oxidants.

J Y Masson 1, D Ramotar 1
PMCID: PMC231196  PMID: 8628275

Abstract

Bleomycin belongs to a class of antitumor drugs that damage cellular DNA through the production of free radicals. The molecular basis by which eukaryotic cells provide resistance to the lethal effects of bleomycin is not clear. Using the yeast Saccharomyces cerevisiae as a model with which to study the effect of bleomycin damage on cellular DNA, we isolated several mutants that display hypersensitivity to bleomycin. A DNA clone containing the IMP2 gene that complemented the most sensitive bleomycin mutant was identified. A role for IMP2 in defense against the toxic effects of bleomycin has not been previously reported. imp2 null mutants were constructed and were found to be 15-fold more sensitive to bleomycin than wild-type strains. The imp2 null mutants were also hypersensitive to several oxidants but displayed parental resistance to UV light and methyl methane sulfonate. Exposure of mutants to either bleomycin or hydrogen peroxide resulted in the accumulation of strand breaks in the chromosomal DNA, which remained even after 6 h postchallenge, but not in the wild type. These results suggest that the oxidant hypersensitivity of the imp2 mutant results from a defect in the repair of oxidative DNA lesions. Molecular analysis of IMP2 indicates that it encodes a transcriptional activator that can activate a reporter gene via an acidic domain located at the N terminus. Imp2 lacks a DNA binding motif, but it possesses a C-terminal leucine-rich repeat. With these data taken together, we propose that Imp2 prevents oxidative damage by regulating the expression of genes that are directly required to repair DNA damage.

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Selected References

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  1. Absalon M. J., Kozarich J. W., Stubbe J. Sequence-specific double-strand cleavage of DNA by Fe-bleomycin. 1. The detection of sequence-specific double-strand breaks using hairpin oligonucleotides. Biochemistry. 1995 Feb 14;34(6):2065–2075. doi: 10.1021/bi00006a029. [DOI] [PubMed] [Google Scholar]
  2. Aguilera A., Klein H. L. Genetic control of intrachromosomal recombination in Saccharomyces cerevisiae. I. Isolation and genetic characterization of hyper-recombination mutations. Genetics. 1988 Aug;119(4):779–790. doi: 10.1093/genetics/119.4.779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Averbeck D., Averbeck S. Induction of the genes RAD54 and RNR2 by various DNA damaging agents in Saccharomyces cerevisiae. Mutat Res. 1994 Sep;315(2):123–138. doi: 10.1016/0921-8777(94)90013-2. [DOI] [PubMed] [Google Scholar]
  4. Bennett R. A., Swerdlow P. S., Povirk L. F. Spontaneous cleavage of bleomycin-induced abasic sites in chromatin and their mutagenicity in mammalian shuttle vectors. Biochemistry. 1993 Mar 30;32(12):3188–3195. doi: 10.1021/bi00063a034. [DOI] [PubMed] [Google Scholar]
  5. Budd M. E., Campbell J. L. DNA polymerases required for repair of UV-induced damage in Saccharomyces cerevisiae. Mol Cell Biol. 1995 Apr;15(4):2173–2179. doi: 10.1128/mcb.15.4.2173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Burger R. M., Drlica K., Birdsall B. The DNA cleavage pathway of iron bleomycin. Strand scission precedes deoxyribose 3-phosphate bond cleavage. J Biol Chem. 1994 Oct 21;269(42):25978–25985. [PubMed] [Google Scholar]
  7. Cunningham R. P., Saporito S. M., Spitzer S. G., Weiss B. Endonuclease IV (nfo) mutant of Escherichia coli. J Bacteriol. 1986 Dec;168(3):1120–1127. doi: 10.1128/jb.168.3.1120-1127.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Demple B., Harrison L. Repair of oxidative damage to DNA: enzymology and biology. Annu Rev Biochem. 1994;63:915–948. doi: 10.1146/annurev.bi.63.070194.004411. [DOI] [PubMed] [Google Scholar]
  9. Demple B., Johnson A., Fung D. Exonuclease III and endonuclease IV remove 3' blocks from DNA synthesis primers in H2O2-damaged Escherichia coli. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7731–7735. doi: 10.1073/pnas.83.20.7731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Donnini C., Lodi T., Ferrero I., Puglisi P. P. IMP2, a nuclear gene controlling the mitochondrial dependence of galactose, maltose and raffinose utilization in Saccharomyces cerevisiae. Yeast. 1992 Feb;8(2):83–93. doi: 10.1002/yea.320080203. [DOI] [PubMed] [Google Scholar]
  11. Draper M. P., Liu H. Y., Nelsbach A. H., Mosley S. P., Denis C. L. CCR4 is a glucose-regulated transcription factor whose leucine-rich repeat binds several proteins important for placing CCR4 in its proper promoter context. Mol Cell Biol. 1994 Jul;14(7):4522–4531. doi: 10.1128/mcb.14.7.4522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Enenkel C., Wolf D. H. BLH1 codes for a yeast thiol aminopeptidase, the equivalent of mammalian bleomycin hydrolase. J Biol Chem. 1993 Apr 5;268(10):7036–7043. [PubMed] [Google Scholar]
  13. Game J. C. DNA double-strand breaks and the RAD50-RAD57 genes in Saccharomyces. Semin Cancer Biol. 1993 Apr;4(2):73–83. [PubMed] [Google Scholar]
  14. Gershoni J. M., Palade G. E. Protein blotting: principles and applications. Anal Biochem. 1983 May;131(1):1–15. doi: 10.1016/0003-2697(83)90128-8. [DOI] [PubMed] [Google Scholar]
  15. Gietz R. D., Schiestl R. H. Applications of high efficiency lithium acetate transformation of intact yeast cells using single-stranded nucleic acids as carrier. Yeast. 1991 Apr;7(3):253–263. doi: 10.1002/yea.320070307. [DOI] [PubMed] [Google Scholar]
  16. Giloni L., Takeshita M., Johnson F., Iden C., Grollman A. P. Bleomycin-induced strand-scission of DNA. Mechanism of deoxyribose cleavage. J Biol Chem. 1981 Aug 25;256(16):8608–8615. [PubMed] [Google Scholar]
  17. Gstaiger M., Knoepfel L., Georgiev O., Schaffner W., Hovens C. M. A B-cell coactivator of octamer-binding transcription factors. Nature. 1995 Jan 26;373(6512):360–362. doi: 10.1038/373360a0. [DOI] [PubMed] [Google Scholar]
  18. Henner W. D., Grunberg S. M., Haseltine W. A. Enzyme action at 3' termini of ionizing radiation-induced DNA strand breaks. J Biol Chem. 1983 Dec 25;258(24):15198–15205. [PubMed] [Google Scholar]
  19. Hope I. A., Mahadevan S., Struhl K. Structural and functional characterization of the short acidic transcriptional activation region of yeast GCN4 protein. Nature. 1988 Jun 16;333(6174):635–640. doi: 10.1038/333635a0. [DOI] [PubMed] [Google Scholar]
  20. Huang K. N., Symington L. S. Mutation of the gene encoding protein kinase C 1 stimulates mitotic recombination in Saccharomyces cerevisiae. Mol Cell Biol. 1994 Sep;14(9):6039–6045. doi: 10.1128/mcb.14.9.6039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Keszenman D. J., Salvo V. A., Nunes E. Effects of bleomycin on growth kinetics and survival of Saccharomyces cerevisiae: a model of repair pathways. J Bacteriol. 1992 May;174(10):3125–3132. doi: 10.1128/jb.174.10.3125-3132.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Khavari P. A., Peterson C. L., Tamkun J. W., Mendel D. B., Crabtree G. R. BRG1 contains a conserved domain of the SWI2/SNF2 family necessary for normal mitotic growth and transcription. Nature. 1993 Nov 11;366(6451):170–174. doi: 10.1038/366170a0. [DOI] [PubMed] [Google Scholar]
  23. Kobe B., Deisenhofer J. A structural basis of the interactions between leucine-rich repeats and protein ligands. Nature. 1995 Mar 9;374(6518):183–186. doi: 10.1038/374183a0. [DOI] [PubMed] [Google Scholar]
  24. Kobe B., Deisenhofer J. The leucine-rich repeat: a versatile binding motif. Trends Biochem Sci. 1994 Oct;19(10):415–421. doi: 10.1016/0968-0004(94)90090-6. [DOI] [PubMed] [Google Scholar]
  25. Kolodner R. D. Mismatch repair: mechanisms and relationship to cancer susceptibility. Trends Biochem Sci. 1995 Oct;20(10):397–401. doi: 10.1016/s0968-0004(00)89087-8. [DOI] [PubMed] [Google Scholar]
  26. Kuge S., Jones N. YAP1 dependent activation of TRX2 is essential for the response of Saccharomyces cerevisiae to oxidative stress by hydroperoxides. EMBO J. 1994 Feb 1;13(3):655–664. doi: 10.1002/j.1460-2075.1994.tb06304.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kunz B. A., Henson E. S., Roche H., Ramotar D., Nunoshiba T., Demple B. Specificity of the mutator caused by deletion of the yeast structural gene (APN1) for the major apurinic endonuclease. Proc Natl Acad Sci U S A. 1994 Aug 16;91(17):8165–8169. doi: 10.1073/pnas.91.17.8165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Laurent B. C., Treitel M. A., Carlson M. The SNF5 protein of Saccharomyces cerevisiae is a glutamine- and proline-rich transcriptional activator that affects expression of a broad spectrum of genes. Mol Cell Biol. 1990 Nov;10(11):5616–5625. doi: 10.1128/mcb.10.11.5616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Levin J. D., Johnson A. W., Demple B. Homogeneous Escherichia coli endonuclease IV. Characterization of an enzyme that recognizes oxidative damage in DNA. J Biol Chem. 1988 Jun 15;263(17):8066–8071. [PubMed] [Google Scholar]
  30. Liang P., Zhu W., Zhang X., Guo Z., O'Connell R. P., Averboukh L., Wang F., Pardee A. B. Differential display using one-base anchored oligo-dT primers. Nucleic Acids Res. 1994 Dec 25;22(25):5763–5764. doi: 10.1093/nar/22.25.5763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lin S. J., Culotta V. C. The ATX1 gene of Saccharomyces cerevisiae encodes a small metal homeostasis factor that protects cells against reactive oxygen toxicity. Proc Natl Acad Sci U S A. 1995 Apr 25;92(9):3784–3788. doi: 10.1073/pnas.92.9.3784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Loeb L. A., Preston B. D. Mutagenesis by apurinic/apyrimidinic sites. Annu Rev Genet. 1986;20:201–230. doi: 10.1146/annurev.ge.20.120186.001221. [DOI] [PubMed] [Google Scholar]
  33. Ma J., Ptashne M. A new class of yeast transcriptional activators. Cell. 1987 Oct 9;51(1):113–119. doi: 10.1016/0092-8674(87)90015-8. [DOI] [PubMed] [Google Scholar]
  34. Magdolen U., Müller G., Magdolen V., Bandlow W. A yeast gene (BLH1) encodes a polypeptide with high homology to vertebrate bleomycin hydrolase, a family member of thiol proteinases. Biochim Biophys Acta. 1993 Jan 23;1171(3):299–303. doi: 10.1016/0167-4781(93)90069-p. [DOI] [PubMed] [Google Scholar]
  35. Moore C. W. Further characterizations of bleomycin-sensitive (blm) mutants of Saccharomyces cerevisiae with implications for a radiomimetic model. J Bacteriol. 1991 Jun;173(11):3605–3608. doi: 10.1128/jb.173.11.3605-3608.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Popoff S. C., Spira A. I., Johnson A. W., Demple B. Yeast structural gene (APN1) for the major apurinic endonuclease: homology to Escherichia coli endonuclease IV. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4193–4197. doi: 10.1073/pnas.87.11.4193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Prakash S., Sung P., Prakash L. DNA repair genes and proteins of Saccharomyces cerevisiae. Annu Rev Genet. 1993;27:33–70. doi: 10.1146/annurev.ge.27.120193.000341. [DOI] [PubMed] [Google Scholar]
  38. Rabindran S. K., Haroun R. I., Clos J., Wisniewski J., Wu C. Regulation of heat shock factor trimer formation: role of a conserved leucine zipper. Science. 1993 Jan 8;259(5092):230–234. doi: 10.1126/science.8421783. [DOI] [PubMed] [Google Scholar]
  39. Ramotar D., Popoff S. C., Demple B. Complementation of DNA repair-deficient Escherichia coli by the yeast Apn1 apurinic/apyrimidinic endonuclease gene. Mol Microbiol. 1991 Jan;5(1):149–155. doi: 10.1111/j.1365-2958.1991.tb01835.x. [DOI] [PubMed] [Google Scholar]
  40. Ramotar D., Popoff S. C., Gralla E. B., Demple B. Cellular role of yeast Apn1 apurinic endonuclease/3'-diesterase: repair of oxidative and alkylation DNA damage and control of spontaneous mutation. Mol Cell Biol. 1991 Sep;11(9):4537–4544. doi: 10.1128/mcb.11.9.4537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ramotar D. Rapid isolation of any known genes from whole cells of yeast by PCR. Mol Cell Biochem. 1995 Apr 26;145(2):185–187. doi: 10.1007/BF00935491. [DOI] [PubMed] [Google Scholar]
  42. Reynolds R. J. Removal of pyrimidine dimers from Saccharomyces cerevisiae nuclear DNA under nongrowth conditions as detected by a sensitive, enzymatic assay. Mutat Res. 1978 Apr;50(1):43–56. doi: 10.1016/0027-5107(78)90059-3. [DOI] [PubMed] [Google Scholar]
  43. Rose M. D. Isolation of genes by complementation in yeast. Methods Enzymol. 1987;152:481–504. doi: 10.1016/0076-6879(87)52056-0. [DOI] [PubMed] [Google Scholar]
  44. Rothstein R. Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. Methods Enzymol. 1991;194:281–301. doi: 10.1016/0076-6879(91)94022-5. [DOI] [PubMed] [Google Scholar]
  45. Ruden D. M., Ma J., Li Y., Wood K., Ptashne M. Generating yeast transcriptional activators containing no yeast protein sequences. Nature. 1991 Mar 21;350(6315):250–252. doi: 10.1038/350250a0. [DOI] [PubMed] [Google Scholar]
  46. Sadowski I., Ma J., Triezenberg S., Ptashne M. GAL4-VP16 is an unusually potent transcriptional activator. Nature. 1988 Oct 6;335(6190):563–564. doi: 10.1038/335563a0. [DOI] [PubMed] [Google Scholar]
  47. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Schmitt M. E., Brown T. A., Trumpower B. L. A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. Nucleic Acids Res. 1990 May 25;18(10):3091–3092. doi: 10.1093/nar/18.10.3091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Seki S., Akiyama K., Watanabe S., Hatsushika M., Ikeda S., Tsutsui K. cDNA and deduced amino acid sequence of a mouse DNA repair enzyme (APEX nuclease) with significant homology to Escherichia coli exonuclease III. J Biol Chem. 1991 Nov 5;266(31):20797–20802. [PubMed] [Google Scholar]
  50. Steighner R. J., Povirk L. F. Bleomycin-induced DNA lesions at mutational hot spots: implications for the mechanism of double-strand cleavage. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8350–8354. doi: 10.1073/pnas.87.21.8350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Szostak J. W., Orr-Weaver T. L., Rothstein R. J., Stahl F. W. The double-strand-break repair model for recombination. Cell. 1983 May;33(1):25–35. doi: 10.1016/0092-8674(83)90331-8. [DOI] [PubMed] [Google Scholar]
  52. Tanaka K., Wood R. D. Xeroderma pigmentosum and nucleotide excision repair of DNA. Trends Biochem Sci. 1994 Feb;19(2):83–86. doi: 10.1016/0968-0004(94)90040-X. [DOI] [PubMed] [Google Scholar]
  53. Umezawa H., Maeda K., Takeuchi T., Okami Y. New antibiotics, bleomycin A and B. J Antibiot (Tokyo) 1966 Sep;19(5):200–209. [PubMed] [Google Scholar]
  54. Worth L., Jr, Frank B. L., Christner D. F., Absalon M. J., Stubbe J., Kozarich J. W. Isotope effects on the cleavage of DNA by bleomycin: mechanism and modulation. Biochemistry. 1993 Mar 16;32(10):2601–2609. doi: 10.1021/bi00061a018. [DOI] [PubMed] [Google Scholar]
  55. Wu A., Wemmie J. A., Edgington N. P., Goebl M., Guevara J. L., Moye-Rowley W. S. Yeast bZip proteins mediate pleiotropic drug and metal resistance. J Biol Chem. 1993 Sep 5;268(25):18850–18858. [PubMed] [Google Scholar]

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