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. 1994 Dec;14(12):8315–8321. doi: 10.1128/mcb.14.12.8315

Distinct residues of human p53 implicated in binding to DNA, simian virus 40 large T antigen, 53BP1, and 53BP2.

S K Thukral 1, G C Blain 1, K K Chang 1, S Fields 1
PMCID: PMC359370  PMID: 7969167

Abstract

We identified a minimal domain of human p53 required for the transactivation of a p53 response element in Saccharomyces cerevisiae. This domain contains the central region of p53 sufficient for specific DNA binding, which colocalizes with the region responsible for binding simian virus 40 large T antigen, 53BP1, and 53BP2. Thirty amino acid positions, including natural mutational hot spots (R175, R213, R248, R249, and R273), in the minimal DNA-binding domain were mutated by alanine substitution. Alanine substitutions at positions R213, R248, R249, D281, R282, R283, E286, and N288 affected transactivation but allowed binding to at least one of the three interacting proteins; these amino acids may be involved in amino acid-base pair contacts. Surprisingly, alanine substitution at the mutational hot spot R175 did not affect DNA binding, transactivation, or T-antigen binding, although it nearly eliminated binding to 53BP1 and 53BP2. Mutation of H168 significantly affected only T-antigen binding, and mutation of E285 affected only 53BP1 binding. Thus, we implicate specific residues of p53 in different DNA and protein interactions.

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Selected References

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

  1. Bargonetti J., Friedman P. N., Kern S. E., Vogelstein B., Prives C. Wild-type but not mutant p53 immunopurified proteins bind to sequences adjacent to the SV40 origin of replication. Cell. 1991 Jun 14;65(6):1083–1091. doi: 10.1016/0092-8674(91)90560-l. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Bargonetti J., Reynisdóttir I., Friedman P. N., Prives C. Site-specific binding of wild-type p53 to cellular DNA is inhibited by SV40 T antigen and mutant p53. Genes Dev. 1992 Oct;6(10):1886–1898. doi: 10.1101/gad.6.10.1886. [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. Dittmer D., Pati S., Zambetti G., Chu S., Teresky A. K., Moore M., Finlay C., Levine A. J. Gain of function mutations in p53. Nat Genet. 1993 May;4(1):42–46. doi: 10.1038/ng0593-42. [DOI] [PubMed] [Google Scholar]
  6. Ebright R. H. Identification of amino acid-base pair contacts by genetic methods. Methods Enzymol. 1991;208:620–640. doi: 10.1016/0076-6879(91)08032-d. [DOI] [PubMed] [Google Scholar]
  7. Farmer G., Bargonetti J., Zhu H., Friedman P., Prywes R., Prives C. Wild-type p53 activates transcription in vitro. Nature. 1992 Jul 2;358(6381):83–86. doi: 10.1038/358083a0. [DOI] [PubMed] [Google Scholar]
  8. Fields S., Jang S. K. Presence of a potent transcription activating sequence in the p53 protein. Science. 1990 Aug 31;249(4972):1046–1049. doi: 10.1126/science.2144363. [DOI] [PubMed] [Google Scholar]
  9. Fields S., Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989 Jul 20;340(6230):245–246. doi: 10.1038/340245a0. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  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. Halazonetis T. D., Kandil A. N. Conformational shifts propagate from the oligomerization domain of p53 to its tetrameric DNA binding domain and restore DNA binding to select p53 mutants. EMBO J. 1993 Dec 15;12(13):5057–5064. doi: 10.1002/j.1460-2075.1993.tb06199.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hinds P., Finlay C., Levine A. J. Mutation is required to activate the p53 gene for cooperation with the ras oncogene and transformation. J Virol. 1989 Feb;63(2):739–746. doi: 10.1128/jvi.63.2.739-746.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hollstein M., Sidransky D., Vogelstein B., Harris C. C. p53 mutations in human cancers. Science. 1991 Jul 5;253(5015):49–53. doi: 10.1126/science.1905840. [DOI] [PubMed] [Google Scholar]
  16. Iwabuchi K., Bartel P. L., Li B., Marraccino R., Fields S. Two cellular proteins that bind to wild-type but not mutant p53. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):6098–6102. doi: 10.1073/pnas.91.13.6098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Iwabuchi K., Li B., Bartel P., Fields S. Use of the two-hybrid system to identify the domain of p53 involved in oligomerization. Oncogene. 1993 Jun;8(6):1693–1696. [PubMed] [Google Scholar]
  18. 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]
  19. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lane D. P. Cancer. p53, guardian of the genome. Nature. 1992 Jul 2;358(6381):15–16. doi: 10.1038/358015a0. [DOI] [PubMed] [Google Scholar]
  21. Lane D. P., Crawford L. V. T antigen is bound to a host protein in SV40-transformed cells. Nature. 1979 Mar 15;278(5701):261–263. doi: 10.1038/278261a0. [DOI] [PubMed] [Google Scholar]
  22. Levine A. J., Momand J., Finlay C. A. The p53 tumour suppressor gene. Nature. 1991 Jun 6;351(6326):453–456. doi: 10.1038/351453a0. [DOI] [PubMed] [Google Scholar]
  23. Linzer D. I., Levine A. J. Characterization of a 54K dalton cellular SV40 tumor antigen present in SV40-transformed cells and uninfected embryonal carcinoma cells. Cell. 1979 May;17(1):43–52. doi: 10.1016/0092-8674(79)90293-9. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Milner J., Medcalf E. A., Cook A. C. Tumor suppressor p53: analysis of wild-type and mutant p53 complexes. Mol Cell Biol. 1991 Jan;11(1):12–19. doi: 10.1128/mcb.11.1.12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nigro J. M., Baker S. J., Preisinger A. C., Jessup J. M., Hostetter R., Cleary K., Bigner S. H., Davidson N., Baylin S., Devilee P. Mutations in the p53 gene occur in diverse human tumour types. Nature. 1989 Dec 7;342(6250):705–708. doi: 10.1038/342705a0. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Prives C., Manfredi J. J. The p53 tumor suppressor protein: meeting review. Genes Dev. 1993 Apr;7(4):529–534. doi: 10.1101/gad.7.4.529. [DOI] [PubMed] [Google Scholar]
  29. Raycroft L., Schmidt J. R., Yoas K., Hao M. M., Lozano G. Analysis of p53 mutants for transcriptional activity. Mol Cell Biol. 1991 Dec;11(12):6067–6074. doi: 10.1128/mcb.11.12.6067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ruppert J. M., Stillman B. Analysis of a protein-binding domain of p53. Mol Cell Biol. 1993 Jun;13(6):3811–3820. doi: 10.1128/mcb.13.6.3811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Schärer E., Iggo R. Mammalian p53 can function as a transcription factor in yeast. Nucleic Acids Res. 1992 Apr 11;20(7):1539–1545. doi: 10.1093/nar/20.7.1539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Shaulsky G., Goldfinger N., Ben-Ze'ev A., Rotter V. Nuclear accumulation of p53 protein is mediated by several nuclear localization signals and plays a role in tumorigenesis. Mol Cell Biol. 1990 Dec;10(12):6565–6577. doi: 10.1128/mcb.10.12.6565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Shaulsky G., Goldfinger N., Tosky M. S., Levine A. J., Rotter V. Nuclear localization is essential for the activity of p53 protein. Oncogene. 1991 Nov;6(11):2055–2065. [PubMed] [Google Scholar]
  34. Stürzbecher H. W., Brain R., Addison C., Rudge K., Remm M., Grimaldi M., Keenan E., Jenkins J. R. A C-terminal alpha-helix plus basic region motif is the major structural determinant of p53 tetramerization. Oncogene. 1992 Aug;7(8):1513–1523. [PubMed] [Google Scholar]
  35. Thukral S. K., Morrison M. L., Young E. T. Mutations in the zinc fingers of ADR1 that change the specificity of DNA binding and transactivation. Mol Cell Biol. 1992 Jun;12(6):2784–2792. doi: 10.1128/mcb.12.6.2784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Unger T., Nau M. M., Segal S., Minna J. D. p53: a transdominant regulator of transcription whose function is ablated by mutations occurring in human cancer. EMBO J. 1992 Apr;11(4):1383–1390. doi: 10.1002/j.1460-2075.1992.tb05183.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Vogelstein B., Kinzler K. W. p53 function and dysfunction. Cell. 1992 Aug 21;70(4):523–526. doi: 10.1016/0092-8674(92)90421-8. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Weintraub H., Hauschka S., Tapscott S. J. The MCK enhancer contains a p53 responsive element. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4570–4571. doi: 10.1073/pnas.88.11.4570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. Methods Enzymol. 1987;154:329–350. doi: 10.1016/0076-6879(87)54083-6. [DOI] [PubMed] [Google Scholar]
  42. 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]

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