Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1997 Mar 15;25(6):1289–1295. doi: 10.1093/nar/25.6.1289

Redox state regulates binding of p53 to sequence-specific DNA, but not to non-specific or mismatched DNA.

D Parks 1, R Bolinger 1, K Mann 1
PMCID: PMC146562  PMID: 9092641

Abstract

Redox modulation of wild-type p53 plays a role in sequence-specific DNA binding in vitro . Reduction produces a DNA-binding form of the protein while oxidation produces a non-DNA-binding form. Primer extension analysis reveals that increasing concentrations of reduced p53 result in enhanced protection of the consensus sequence, while increasing concentrations of oxidized p53 confer minimal protection of the consensus sequence. DNA binding by oxidized p53 is, therefore, not sequence-specific. In contrast, there is no observable difference in the binding of oxidized p53 and reduced p53 to double-stranded non-specific or mismatched DNA in gel mobility shift assays. Both forms of p53 bind equally well, suggesting that redox modulation of p53 does not play a role in its binding to non-specific or mismatched DNA. In view of the in vitro evidence that redox state influences the sequence-specific DNA-binding of p53, we have examined the effect of oxidative stress on the in vivo ability of p53 to bind to and transactivate PG13-CAT, a reporter construct containing multiple copies of the p53 consensus binding site linked to the chloramphenicol acetyltransferase gene. Hydrogen peroxide treatment of cells cotransfected with p53 results in a marked decrease in CAT activity, suggesting that oxidation of p53 decreases the ability of the protein to bind to consensus DNA and transactivate target genes in vivo.

Full Text

The Full Text of this article is available as a PDF (166.9 KB).

Selected References

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

  1. Abate C., Patel L., Rauscher F. J., 3rd, Curran T. Redox regulation of fos and jun DNA-binding activity in vitro. Science. 1990 Sep 7;249(4973):1157–1161. doi: 10.1126/science.2118682. [DOI] [PubMed] [Google Scholar]
  2. Bakalkin G., Yakovleva T., Selivanova G., Magnusson K. P., Szekely L., Kiseleva E., Klein G., Terenius L., Wiman K. G. p53 binds single-stranded DNA ends and catalyzes DNA renaturation and strand transfer. Proc Natl Acad Sci U S A. 1994 Jan 4;91(1):413–417. doi: 10.1073/pnas.91.1.413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bargonetti J., Manfredi J. J., Chen X., Marshak D. R., Prives C. A proteolytic fragment from the central region of p53 has marked sequence-specific DNA-binding activity when generated from wild-type but not from oncogenic mutant p53 protein. Genes Dev. 1993 Dec;7(12B):2565–2574. doi: 10.1101/gad.7.12b.2565. [DOI] [PubMed] [Google Scholar]
  4. Cho Y., Gorina S., Jeffrey P. D., Pavletich N. P. Crystal structure of a p53 tumor suppressor-DNA complex: understanding tumorigenic mutations. Science. 1994 Jul 15;265(5170):346–355. doi: 10.1126/science.8023157. [DOI] [PubMed] [Google Scholar]
  5. Clarke A. R., Purdie C. A., Harrison D. J., Morris R. G., Bird C. C., Hooper M. L., Wyllie A. H. Thymocyte apoptosis induced by p53-dependent and independent pathways. Nature. 1993 Apr 29;362(6423):849–852. doi: 10.1038/362849a0. [DOI] [PubMed] [Google Scholar]
  6. Cox L. S., Hupp T., Midgley C. A., Lane D. P. A direct effect of activated human p53 on nuclear DNA replication. EMBO J. 1995 May 1;14(9):2099–2105. doi: 10.1002/j.1460-2075.1995.tb07201.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Devary Y., Gottlieb R. A., Lau L. F., Karin M. Rapid and preferential activation of the c-jun gene during the mammalian UV response. Mol Cell Biol. 1991 May;11(5):2804–2811. doi: 10.1128/mcb.11.5.2804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Finlay C. A., Hinds P. W., Levine A. J. The p53 proto-oncogene can act as a suppressor of transformation. Cell. 1989 Jun 30;57(7):1083–1093. doi: 10.1016/0092-8674(89)90045-7. [DOI] [PubMed] [Google Scholar]
  9. Fritsche M., Haessler C., Brandner G. Induction of nuclear accumulation of the tumor-suppressor protein p53 by DNA-damaging agents. Oncogene. 1993 Feb;8(2):307–318. [PubMed] [Google Scholar]
  10. Funk W. D., Pak D. T., Karas R. H., Wright W. E., Shay J. W. A transcriptionally active DNA-binding site for human p53 protein complexes. Mol Cell Biol. 1992 Jun;12(6):2866–2871. doi: 10.1128/mcb.12.6.2866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gibbs J. B. Ras C-terminal processing enzymes--new drug targets? Cell. 1991 Apr 5;65(1):1–4. doi: 10.1016/0092-8674(91)90352-y. [DOI] [PubMed] [Google Scholar]
  12. Graeber T. G., Osmanian C., Jacks T., Housman D. E., Koch C. J., Lowe S. W., Giaccia A. J. Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours. Nature. 1996 Jan 4;379(6560):88–91. doi: 10.1038/379088a0. [DOI] [PubMed] [Google Scholar]
  13. Graeber T. G., Peterson J. F., Tsai M., Monica K., Fornace A. J., Jr, Giaccia A. J. Hypoxia induces accumulation of p53 protein, but activation of a G1-phase checkpoint by low-oxygen conditions is independent of p53 status. Mol Cell Biol. 1994 Sep;14(9):6264–6277. doi: 10.1128/mcb.14.9.6264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gralla J. D. Rapid "footprinting" on supercoiled DNA. Proc Natl Acad Sci U S A. 1985 May;82(10):3078–3081. doi: 10.1073/pnas.82.10.3078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hainaut P., Milner J. A structural role for metal ions in the "wild-type" conformation of the tumor suppressor protein p53. Cancer Res. 1993 Apr 15;53(8):1739–1742. [PubMed] [Google Scholar]
  16. Hainaut P., Milner J. Redox modulation of p53 conformation and sequence-specific DNA binding in vitro. Cancer Res. 1993 Oct 1;53(19):4469–4473. [PubMed] [Google Scholar]
  17. Harper J. W., Adami G. R., Wei N., Keyomarsi K., Elledge S. J. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell. 1993 Nov 19;75(4):805–816. doi: 10.1016/0092-8674(93)90499-g. [DOI] [PubMed] [Google Scholar]
  18. Hidalgo E., Demple B. An iron-sulfur center essential for transcriptional activation by the redox-sensing SoxR protein. EMBO J. 1994 Jan 1;13(1):138–146. doi: 10.1002/j.1460-2075.1994.tb06243.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hupp T. R., Meek D. W., Midgley C. A., Lane D. P. Activation of the cryptic DNA binding function of mutant forms of p53. Nucleic Acids Res. 1993 Jul 11;21(14):3167–3174. doi: 10.1093/nar/21.14.3167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hupp T. R., Meek D. W., Midgley C. A., Lane D. P. Regulation of the specific DNA binding function of p53. Cell. 1992 Nov 27;71(5):875–886. doi: 10.1016/0092-8674(92)90562-q. [DOI] [PubMed] [Google Scholar]
  21. Jayaraman J., Prives C. Activation of p53 sequence-specific DNA binding by short single strands of DNA requires the p53 C-terminus. Cell. 1995 Jun 30;81(7):1021–1029. doi: 10.1016/s0092-8674(05)80007-8. [DOI] [PubMed] [Google Scholar]
  22. Kastan M. B., Zhan Q., el-Deiry W. S., Carrier F., Jacks T., Walsh W. V., Plunkett B. S., Vogelstein B., Fornace A. J., Jr A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell. 1992 Nov 13;71(4):587–597. doi: 10.1016/0092-8674(92)90593-2. [DOI] [PubMed] [Google Scholar]
  23. Kern S. E., Pietenpol J. A., Thiagalingam S., Seymour A., Kinzler K. W., Vogelstein B. Oncogenic forms of p53 inhibit p53-regulated gene expression. Science. 1992 May 8;256(5058):827–830. doi: 10.1126/science.1589764. [DOI] [PubMed] [Google Scholar]
  24. Lane D. P., Stephen C. W., Midgley C. A., Sparks A., Hupp T. R., Daniels D. A., Greaves R., Reid A., Vojtesek B., Picksley S. M. Epitope analysis of the murine p53 tumour suppressor protein. Oncogene. 1996 Jun 6;12(11):2461–2466. [PubMed] [Google Scholar]
  25. Lee S., Elenbaas B., Levine A., Griffith J. p53 and its 14 kDa C-terminal domain recognize primary DNA damage in the form of insertion/deletion mismatches. Cell. 1995 Jun 30;81(7):1013–1020. doi: 10.1016/s0092-8674(05)80006-6. [DOI] [PubMed] [Google Scholar]
  26. Lowe S. W., Schmitt E. M., Smith S. W., Osborne B. A., Jacks T. p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature. 1993 Apr 29;362(6423):847–849. doi: 10.1038/362847a0. [DOI] [PubMed] [Google Scholar]
  27. Lu X., Lane D. P. Differential induction of transcriptionally active p53 following UV or ionizing radiation: defects in chromosome instability syndromes? Cell. 1993 Nov 19;75(4):765–778. doi: 10.1016/0092-8674(93)90496-d. [DOI] [PubMed] [Google Scholar]
  28. Meyer M., Schreck R., Baeuerle P. A. H2O2 and antioxidants have opposite effects on activation of NF-kappa B and AP-1 in intact cells: AP-1 as secondary antioxidant-responsive factor. EMBO J. 1993 May;12(5):2005–2015. doi: 10.1002/j.1460-2075.1993.tb05850.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Miller S. D., Farmer G., Prives C. p53 inhibits DNA replication in vitro in a DNA-binding-dependent manner. Mol Cell Biol. 1995 Dec;15(12):6554–6560. doi: 10.1128/mcb.15.12.6554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Miyashita T., Reed J. C. Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell. 1995 Jan 27;80(2):293–299. doi: 10.1016/0092-8674(95)90412-3. [DOI] [PubMed] [Google Scholar]
  31. Mosner J., Mummenbrauer T., Bauer C., Sczakiel G., Grosse F., Deppert W. Negative feedback regulation of wild-type p53 biosynthesis. EMBO J. 1995 Sep 15;14(18):4442–4449. doi: 10.1002/j.1460-2075.1995.tb00123.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Oberosler P., Hloch P., Ramsperger U., Stahl H. p53-catalyzed annealing of complementary single-stranded nucleic acids. EMBO J. 1993 Jun;12(6):2389–2396. doi: 10.1002/j.1460-2075.1993.tb05893.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Parsons R., Anderson M. E., Tegtmeyer P. Three domains in the simian virus 40 core origin orchestrate the binding, melting, and DNA helicase activities of T antigen. J Virol. 1990 Feb;64(2):509–518. doi: 10.1128/jvi.64.2.509-518.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Pavletich N. P., Chambers K. A., Pabo C. O. The DNA-binding domain of p53 contains the four conserved regions and the major mutation hot spots. Genes Dev. 1993 Dec;7(12B):2556–2564. doi: 10.1101/gad.7.12b.2556. [DOI] [PubMed] [Google Scholar]
  35. Rainwater R., Parks D., Anderson M. E., Tegtmeyer P., Mann K. Role of cysteine residues in regulation of p53 function. Mol Cell Biol. 1995 Jul;15(7):3892–3903. doi: 10.1128/mcb.15.7.3892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Reed M., Woelker B., Wang P., Wang Y., Anderson M. E., Tegtmeyer P. The C-terminal domain of p53 recognizes DNA damaged by ionizing radiation. Proc Natl Acad Sci U S A. 1995 Oct 10;92(21):9455–9459. doi: 10.1073/pnas.92.21.9455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Schreck R., Rieber P., Baeuerle P. A. Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kappa B transcription factor and HIV-1. EMBO J. 1991 Aug;10(8):2247–2258. doi: 10.1002/j.1460-2075.1991.tb07761.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Stenger J. E., Mayr G. A., Mann K., Tegtmeyer P. Formation of stable p53 homotetramers and multiples of tetramers. Mol Carcinog. 1992;5(2):102–106. doi: 10.1002/mc.2940050204. [DOI] [PubMed] [Google Scholar]
  39. Sun X., Shimizu H., Yamamoto K. Identification of a novel p53 promoter element involved in genotoxic stress-inducible p53 gene expression. Mol Cell Biol. 1995 Aug;15(8):4489–4496. doi: 10.1128/mcb.15.8.4489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sun Y., Oberley L. W. Redox regulation of transcriptional activators. Free Radic Biol Med. 1996;21(3):335–348. doi: 10.1016/0891-5849(96)00109-8. [DOI] [PubMed] [Google Scholar]
  41. Toledano M. B., Kullik I., Trinh F., Baird P. T., Schneider T. D., Storz G. Redox-dependent shift of OxyR-DNA contacts along an extended DNA-binding site: a mechanism for differential promoter selection. Cell. 1994 Sep 9;78(5):897–909. doi: 10.1016/s0092-8674(94)90702-1. [DOI] [PubMed] [Google Scholar]
  42. Walker L. J., Craig R. B., Harris A. L., Hickson I. D. A role for the human DNA repair enzyme HAP1 in cellular protection against DNA damaging agents and hypoxic stress. Nucleic Acids Res. 1994 Nov 25;22(23):4884–4889. doi: 10.1093/nar/22.23.4884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wang Y., Reed M., Wang P., Stenger J. E., Mayr G., Anderson M. E., Schwedes J. F., Tegtmeyer P. p53 domains: identification and characterization of two autonomous DNA-binding regions. Genes Dev. 1993 Dec;7(12B):2575–2586. doi: 10.1101/gad.7.12b.2575. [DOI] [PubMed] [Google Scholar]
  44. Wang Y., Schwedes J. F., Parks D., Mann K., Tegtmeyer P. Interaction of p53 with its consensus DNA-binding site. Mol Cell Biol. 1995 Apr;15(4):2157–2165. doi: 10.1128/mcb.15.4.2157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Xanthoudakis S., Curran T. Identification and characterization of Ref-1, a nuclear protein that facilitates AP-1 DNA-binding activity. EMBO J. 1992 Feb;11(2):653–665. doi: 10.1002/j.1460-2075.1992.tb05097.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Xanthoudakis S., Miao G. G., Curran T. The redox and DNA-repair activities of Ref-1 are encoded by nonoverlapping domains. Proc Natl Acad Sci U S A. 1994 Jan 4;91(1):23–27. doi: 10.1073/pnas.91.1.23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Xanthoudakis S., Miao G., Wang F., Pan Y. C., Curran T. Redox activation of Fos-Jun DNA binding activity is mediated by a DNA repair enzyme. EMBO J. 1992 Sep;11(9):3323–3335. doi: 10.1002/j.1460-2075.1992.tb05411.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Yao K. S., Clayton M., O'Dwyer P. J. Apoptosis in human adenocarcinoma HT29 cells induced by exposure to hypoxia. J Natl Cancer Inst. 1995 Jan 18;87(2):117–122. doi: 10.1093/jnci/87.2.117. [DOI] [PubMed] [Google Scholar]
  49. Yao K. S., Xanthoudakis S., Curran T., O'Dwyer P. J. Activation of AP-1 and of a nuclear redox factor, Ref-1, in the response of HT29 colon cancer cells to hypoxia. Mol Cell Biol. 1994 Sep;14(9):5997–6003. doi: 10.1128/mcb.14.9.5997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Zambetti G. P., Bargonetti J., Walker K., Prives C., Levine A. J. Wild-type p53 mediates positive regulation of gene expression through a specific DNA sequence element. Genes Dev. 1992 Jul;6(7):1143–1152. doi: 10.1101/gad.6.7.1143. [DOI] [PubMed] [Google Scholar]
  51. el-Deiry W. S., Harper J. W., O'Connor P. M., Velculescu V. E., Canman C. E., Jackman J., Pietenpol J. A., Burrell M., Hill D. E., Wang Y. WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis. Cancer Res. 1994 Mar 1;54(5):1169–1174. [PubMed] [Google Scholar]
  52. el-Deiry W. S., Kern S. E., Pietenpol J. A., Kinzler K. W., Vogelstein B. Definition of a consensus binding site for p53. Nat Genet. 1992 Apr;1(1):45–49. doi: 10.1038/ng0492-45. [DOI] [PubMed] [Google Scholar]
  53. el-Deiry W. S., Tokino T., Velculescu V. E., Levy D. B., Parsons R., Trent J. M., Lin D., Mercer W. E., Kinzler K. W., Vogelstein B. WAF1, a potential mediator of p53 tumor suppression. Cell. 1993 Nov 19;75(4):817–825. doi: 10.1016/0092-8674(93)90500-p. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES