Skip to main content
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1991 Dec 15;88(24):11251–11255. doi: 10.1073/pnas.88.24.11251

A damage-responsive DNA binding protein regulates transcription of the yeast DNA repair gene PHR1.

J Sebastian 1, G B Sancar 1
PMCID: PMC53112  PMID: 1763039

Abstract

The PHR1 gene of Saccharomyces cerevisiae encodes the DNA repair enzyme photolyase. Transcription of PHR1 increases in response to treatment of cells with 254-nm radiation and chemical agents that damage DNA. We report here the identification of a damage-responsive DNA binding protein, termed photolyase regulatory protein (PRP), and its cognate binding site, termed the PHR1 upstream repression sequence, that together regulate induction of PHR1 transcription after DNA damage. PRP activity, monitored by electrophoretic-mobility-shift assay, was detected in cells during normal growth but disappeared within 30 min after irradiation. Copper-phenanthroline footprinting of PRP-DNA complexes revealed that PRP protects a 39-base-pair region of PHR1 5' flanking sequence beginning 40 base pairs upstream from the coding sequence. A prominent feature of the foot-printed region is a 22-base-pair palindrome. Deletion of the PHR1 upstream repression sequence increased the basal level expression of PHR1 in vivo and decreased induction after exposure of cells to UV radiation or methyl methanesulfonate, whereas insertion of the PRP binding site between the CYC1 upstream activation sequence and "TATA" sequence reduced basal level expression and conferred damage responsiveness upon a reporter gene. Thus these observations establish that PRP is a damage-responsive repressor of PHR1 transcription.

Full text

PDF
11251

Images in this article

Selected References

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

  1. Brand A. H., Micklem G., Nasmyth K. A yeast silencer contains sequences that can promote autonomous plasmid replication and transcriptional activation. Cell. 1987 Dec 4;51(5):709–719. doi: 10.1016/0092-8674(87)90094-8. [DOI] [PubMed] [Google Scholar]
  2. Cole G. M., Mortimer R. K. Failure to induce a DNA repair gene, RAD54, in Saccharomyces cerevisiae does not affect DNA repair or recombination phenotypes. Mol Cell Biol. 1989 Aug;9(8):3314–3322. doi: 10.1128/mcb.9.8.3314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. DasGupta U. B., Summers W. C. Ultraviolet reactivation of herpes simplex virus is mutagenic and inducible in mammlian cells. Proc Natl Acad Sci U S A. 1978 May;75(5):2378–2381. doi: 10.1073/pnas.75.5.2378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Elledge S. J., Davis R. W. Identification of the DNA damage-responsive element of RNR2 and evidence that four distinct cellular factors bind it. Mol Cell Biol. 1989 Dec;9(12):5373–5386. doi: 10.1128/mcb.9.12.5373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Finley R. L., Jr, Chen S., Ma J., Byrne P., West R. W., Jr Opposing regulatory functions of positive and negative elements in UASG control transcription of the yeast GAL genes. Mol Cell Biol. 1990 Nov;10(11):5663–5670. doi: 10.1128/mcb.10.11.5663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fornace A. J., Jr, Nebert D. W., Hollander M. C., Luethy J. D., Papathanasiou M., Fargnoli J., Holbrook N. J. Mammalian genes coordinately regulated by growth arrest signals and DNA-damaging agents. Mol Cell Biol. 1989 Oct;9(10):4196–4203. doi: 10.1128/mcb.9.10.4196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fried M., Crothers D. M. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Dec 11;9(23):6505–6525. doi: 10.1093/nar/9.23.6505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Garner M. M., Revzin A. A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res. 1981 Jul 10;9(13):3047–3060. doi: 10.1093/nar/9.13.3047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Holliday R. Further evidence for an inducible recombination repair system in Ustilago maydis. Mutat Res. 1975 Jul;29(1):149–153. doi: 10.1016/0027-5107(75)90029-9. [DOI] [PubMed] [Google Scholar]
  10. Hurd H. K., Roberts J. W. Upstream regulatory sequences of the yeast RNR2 gene include a repression sequence and an activation site that binds the RAP1 protein. Mol Cell Biol. 1989 Dec;9(12):5359–5372. doi: 10.1128/mcb.9.12.5359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Johnson A. D., Herskowitz I. A repressor (MAT alpha 2 Product) and its operator control expression of a set of cell type specific genes in yeast. Cell. 1985 Aug;42(1):237–247. doi: 10.1016/s0092-8674(85)80119-7. [DOI] [PubMed] [Google Scholar]
  12. Jones J. S., Prakash L. Transcript levels of the Saccharomyces cerevisiae DNA repair gene RAD18 increase in UV irradiated cells and during meiosis but not during the mitotic cell cycle. Nucleic Acids Res. 1991 Feb 25;19(4):893–898. doi: 10.1093/nar/19.4.893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kuwabara M. D., Sigman D. S. Footprinting DNA-protein complexes in situ following gel retardation assays using 1,10-phenanthroline-copper ion: Escherichia coli RNA polymerase-lac promoter complexes. Biochemistry. 1987 Nov 17;26(23):7234–7238. doi: 10.1021/bi00397a006. [DOI] [PubMed] [Google Scholar]
  14. Kuwabara M., Yoon C., Goyne T., Thederahn T., Sigman D. S. Nuclease activity of 1,10-phenanthroline-copper ion: reaction with CGCGAATTCGCG and its complexes with netropsin and EcoRI. Biochemistry. 1986 Nov 18;25(23):7401–7408. doi: 10.1021/bi00371a023. [DOI] [PubMed] [Google Scholar]
  15. Lalonde B., Arcangioli B., Guarente L. A single Saccharomyces cerevisiae upstream activation site (UAS1) has two distinct regions essential for its activity. Mol Cell Biol. 1986 Dec;6(12):4690–4696. doi: 10.1128/mcb.6.12.4690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lee M. G., Yarranton G. T. Inducible DNA repair in Ustilago maydis. Mol Gen Genet. 1982;185(2):245–250. doi: 10.1007/BF00330793. [DOI] [PubMed] [Google Scholar]
  17. Little J. W., Mount D. W. The SOS regulatory system of Escherichia coli. Cell. 1982 May;29(1):11–22. doi: 10.1016/0092-8674(82)90085-x. [DOI] [PubMed] [Google Scholar]
  18. Mount D. W. A mutant of Escherichia coli showing constitutive expression of the lysogenic induction and error-prone DNA repair pathways. Proc Natl Acad Sci U S A. 1977 Jan;74(1):300–304. doi: 10.1073/pnas.74.1.300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mount D. W., Low K. B., Edmiston S. J. Dominant mutations (lex) in Escherichia coli K-12 which affect radiation sensitivity and frequency of ultraviolet lght-induced mutations. J Bacteriol. 1972 Nov;112(2):886–893. doi: 10.1128/jb.112.2.886-893.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Park H. O., Craig E. A. Positive and negative regulation of basal expression of a yeast HSP70 gene. Mol Cell Biol. 1989 May;9(5):2025–2033. doi: 10.1128/mcb.9.5.2025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pfeifer K., Prezant T., Guarente L. Yeast HAP1 activator binds to two upstream activation sites of different sequence. Cell. 1987 Apr 10;49(1):19–27. doi: 10.1016/0092-8674(87)90751-3. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Sancar G. B. DNA photolyases: physical properties, action mechanism, and roles in dark repair. Mutat Res. 1990 Sep-Nov;236(2-3):147–160. doi: 10.1016/0921-8777(90)90002-m. [DOI] [PubMed] [Google Scholar]
  24. Sancar G. B. Sequence of the Saccharomyces cerevisiae PHR1 gene and homology of the PHR1 photolyase to E. coli photolyase. Nucleic Acids Res. 1985 Nov 25;13(22):8231–8246. doi: 10.1093/nar/13.22.8231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sarasin A., Benoit A. Induction of an error-prone mode of DNA repair in UV-irradiated monkey kidney cells. Mutat Res. 1980 Mar;70(1):71–81. doi: 10.1016/0027-5107(80)90059-7. [DOI] [PubMed] [Google Scholar]
  26. Sebastian J., Kraus B., Sancar G. B. Expression of the yeast PHR1 gene is induced by DNA-damaging agents. Mol Cell Biol. 1990 Sep;10(9):4630–4637. doi: 10.1128/mcb.10.9.4630. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Siede W., Robinson G. W., Kalainov D., Malley T., Friedberg E. C. Regulation of the RAD2 gene of Saccharomyces cerevisiae. Mol Microbiol. 1989 Dec;3(12):1697–1707. doi: 10.1111/j.1365-2958.1989.tb00155.x. [DOI] [PubMed] [Google Scholar]
  28. Stein B., Rahmsdorf H. J., Steffen A., Litfin M., Herrlich P. UV-induced DNA damage is an intermediate step in UV-induced expression of human immunodeficiency virus type 1, collagenase, c-fos, and metallothionein. Mol Cell Biol. 1989 Nov;9(11):5169–5181. doi: 10.1128/mcb.9.11.5169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sumrada R. A., Cooper T. G. Ubiquitous upstream repression sequences control activation of the inducible arginase gene in yeast. Proc Natl Acad Sci U S A. 1987 Jun;84(12):3997–4001. doi: 10.1073/pnas.84.12.3997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Verdier J. M. Regulatory DNA-binding proteins in yeast: an overview. Yeast. 1990 Jul-Aug;6(4):271–297. doi: 10.1002/yea.320060402. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Yagle K., McEntee K. The DNA damage-inducible gene DIN1 of Saccharomyces cerevisiae encodes a regulatory subunit of ribonucleotide reductase and is identical to RNR3. Mol Cell Biol. 1990 Oct;10(10):5553–5557. doi: 10.1128/mcb.10.10.5553. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES