Abstract
Wild-type p53 was shown to function as a transcription factor. The N-terminal region of the protein contains the transcription activation domain, while the C terminus is responsible for DNA binding. Localization of the DNA-binding domain of the p53 protein to the highly conserved carboxy-terminal region suggests that the interaction of p53 with DNA is important for its function. We have developed a strategy for studying the DNA sequence specificity of p53-DNA binding that is based on random sequence selection. We report here on the isolation of murine genomic DNA clones that are specifically bound by the wild-type p53 protein but are not bound by mutant p53 protein forms. The isolated p53 target gene contains the unique DNA-binding sequence GACACTGGTCACACTTGGCTGCTTAGGAAT. This fragment exhibits promoter activity as measured by its capacity to activate transcription of the chloramphenicol acetyltransferase reporter gene. Our results suggest that p53 directly binds DNA and functions as a typical transcription factor.
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- Addison C., Jenkins J. R., Stürzbecher H. W. The p53 nuclear localisation signal is structurally linked to a p34cdc2 kinase motif. Oncogene. 1990 Mar;5(3):423–426. [PubMed] [Google Scholar]
- Ahuja H., Bar-Eli M., Advani S. H., Benchimol S., Cline M. J. Alterations in the p53 gene and the clonal evolution of the blast crisis of chronic myelocytic leukemia. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6783–6787. doi: 10.1073/pnas.86.17.6783. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Arai N., Nomura D., Yokota K., Wolf D., Brill E., Shohat O., Rotter V. Immunologically distinct p53 molecules generated by alternative splicing. Mol Cell Biol. 1986 Sep;6(9):3232–3239. doi: 10.1128/mcb.6.9.3232. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Baker S. J., Fearon E. R., Nigro J. M., Hamilton S. R., Preisinger A. C., Jessup J. M., vanTuinen P., Ledbetter D. H., Barker D. F., Nakamura Y. Chromosome 17 deletions and p53 gene mutations in colorectal carcinomas. Science. 1989 Apr 14;244(4901):217–221. doi: 10.1126/science.2649981. [DOI] [PubMed] [Google Scholar]
- Baker S. J., Markowitz S., Fearon E. R., Willson J. K., Vogelstein B. Suppression of human colorectal carcinoma cell growth by wild-type p53. Science. 1990 Aug 24;249(4971):912–915. doi: 10.1126/science.2144057. [DOI] [PubMed] [Google Scholar]
- 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]
- Bischoff J. R., Casso D., Beach D. Human p53 inhibits growth in Schizosaccharomyces pombe. Mol Cell Biol. 1992 Apr;12(4):1405–1411. doi: 10.1128/mcb.12.4.1405. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Braithwaite A. W., Sturzbecher H. W., Addison C., Palmer C., Rudge K., Jenkins J. R. Mouse p53 inhibits SV40 origin-dependent DNA replication. Nature. 1987 Oct 1;329(6138):458–460. doi: 10.1038/329458a0. [DOI] [PubMed] [Google Scholar]
- Chen P. L., Chen Y. M., Bookstein R., Lee W. H. Genetic mechanisms of tumor suppression by the human p53 gene. Science. 1990 Dec 14;250(4987):1576–1580. doi: 10.1126/science.2274789. [DOI] [PubMed] [Google Scholar]
- Chen Y. M., Chen P. L., Arnaiz N., Goodrich D., Lee W. H. Expression of wild-type p53 in human A673 cells suppresses tumorigenicity but not growth rate. Oncogene. 1991 Oct;6(10):1799–1805. [PubMed] [Google Scholar]
- Chin K. V., Ueda K., Pastan I., Gottesman M. M. Modulation of activity of the promoter of the human MDR1 gene by Ras and p53. Science. 1992 Jan 24;255(5043):459–462. doi: 10.1126/science.1346476. [DOI] [PubMed] [Google Scholar]
- Dang C. V., Lee W. M. Nuclear and nucleolar targeting sequences of c-erb-A, c-myb, N-myc, p53, HSP70, and HIV tat proteins. J Biol Chem. 1989 Oct 25;264(30):18019–18023. [PubMed] [Google Scholar]
- Deppert W., Buschhausen-Denker G., Patschinsky T., Steinmeyer K. Cell cycle control of p53 in normal (3T3) and chemically transformed (Meth A) mouse cells. II. Requirement for cell cycle progression. Oncogene. 1990 Nov;5(11):1701–1706. [PubMed] [Google Scholar]
- Diller L., Kassel J., Nelson C. E., Gryka M. A., Litwak G., Gebhardt M., Bressac B., Ozturk M., Baker S. J., Vogelstein B. p53 functions as a cell cycle control protein in osteosarcomas. Mol Cell Biol. 1990 Nov;10(11):5772–5781. doi: 10.1128/mcb.10.11.5772. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Eliyahu D., Goldfinger N., Pinhasi-Kimhi O., Shaulsky G., Skurnik Y., Arai N., Rotter V., Oren M. Meth A fibrosarcoma cells express two transforming mutant p53 species. Oncogene. 1988 Sep;3(3):313–321. [PubMed] [Google Scholar]
- Eliyahu D., Michalovitz D., Eliyahu S., Pinhasi-Kimhi O., Oren M. Wild-type p53 can inhibit oncogene-mediated focus formation. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8763–8767. doi: 10.1073/pnas.86.22.8763. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Foord O. S., Bhattacharya P., Reich Z., Rotter V. A DNA binding domain is contained in the C-terminus of wild type p53 protein. Nucleic Acids Res. 1991 Oct 11;19(19):5191–5198. doi: 10.1093/nar/19.19.5191. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gannon J. V., Greaves R., Iggo R., Lane D. P. Activating mutations in p53 produce a common conformational effect. A monoclonal antibody specific for the mutant form. EMBO J. 1990 May;9(5):1595–1602. doi: 10.1002/j.1460-2075.1990.tb08279.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gannon J. V., Lane D. P. p53 and DNA polymerase alpha compete for binding to SV40 T antigen. Nature. 1987 Oct 1;329(6138):456–458. doi: 10.1038/329456a0. [DOI] [PubMed] [Google Scholar]
- Ginsberg D., Mechta F., Yaniv M., Oren M. Wild-type p53 can down-modulate the activity of various promoters. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):9979–9983. doi: 10.1073/pnas.88.22.9979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ginsberg D., Michael-Michalovitz D., Ginsberg D., Oren M. Induction of growth arrest by a temperature-sensitive p53 mutant is correlated with increased nuclear localization and decreased stability of the protein. Mol Cell Biol. 1991 Jan;11(1):582–585. doi: 10.1128/mcb.11.1.582. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
- 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]
- Kelman Z., Prokocimer M., Peller S., Kahn Y., Rechavi G., Manor Y., Cohen A., Rotter V. Rearrangements in the p53 gene in Philadelphia chromosome positive chronic myelogenous leukemia. Blood. 1989 Nov 15;74(7):2318–2324. [PubMed] [Google Scholar]
- Kern S. E., Kinzler K. W., Baker S. J., Nigro J. M., Rotter V., Levine A. J., Friedman P., Prives C., Vogelstein B. Mutant p53 proteins bind DNA abnormally in vitro. Oncogene. 1991 Jan;6(1):131–136. [PubMed] [Google Scholar]
- Kern S. E., Kinzler K. W., Bruskin A., Jarosz D., Friedman P., Prives C., Vogelstein B. Identification of p53 as a sequence-specific DNA-binding protein. Science. 1991 Jun 21;252(5013):1708–1711. doi: 10.1126/science.2047879. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Marahrens Y., Stillman B. A yeast chromosomal origin of DNA replication defined by multiple functional elements. Science. 1992 Feb 14;255(5046):817–823. doi: 10.1126/science.1536007. [DOI] [PubMed] [Google Scholar]
- Martinez J., Georgoff I., Martinez J., Levine A. J. Cellular localization and cell cycle regulation by a temperature-sensitive p53 protein. Genes Dev. 1991 Feb;5(2):151–159. doi: 10.1101/gad.5.2.151. [DOI] [PubMed] [Google Scholar]
- Mercer W. E., Shields M. T., Amin M., Sauve G. J., Appella E., Romano J. W., Ullrich S. J. Negative growth regulation in a glioblastoma tumor cell line that conditionally expresses human wild-type p53. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6166–6170. doi: 10.1073/pnas.87.16.6166. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Michalovitz D., Halevy O., Oren M. Conditional inhibition of transformation and of cell proliferation by a temperature-sensitive mutant of p53. Cell. 1990 Aug 24;62(4):671–680. doi: 10.1016/0092-8674(90)90113-s. [DOI] [PubMed] [Google Scholar]
- Mohr I. J., Clark R., Sun S., Androphy E. J., MacPherson P., Botchan M. R. Targeting the E1 replication protein to the papillomavirus origin of replication by complex formation with the E2 transactivator. Science. 1990 Dec 21;250(4988):1694–1699. doi: 10.1126/science.2176744. [DOI] [PubMed] [Google Scholar]
- 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]
- O'Rourke R. W., Miller C. W., Kato G. J., Simon K. J., Chen D. L., Dang C. V., Koeffler H. P. A potential transcriptional activation element in the p53 protein. Oncogene. 1990 Dec;5(12):1829–1832. [PubMed] [Google Scholar]
- Paige C. J., Kincade P. W., Ralph P. Murine B cell leukemia line with inducible surface immunoglobulin expression. J Immunol. 1978 Aug;121(2):641–647. [PubMed] [Google Scholar]
- Prokocimer M., Shaklai M., Bassat H. B., Wolf D., Goldfinger N., Rotter V. Expression of p53 in human leukemia and lymphoma. Blood. 1986 Jul;68(1):113–118. [PubMed] [Google Scholar]
- Raycroft L., Wu H. Y., Lozano G. Transcriptional activation by wild-type but not transforming mutants of the p53 anti-oncogene. Science. 1990 Aug 31;249(4972):1049–1051. doi: 10.1126/science.2144364. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rodrigues N. R., Rowan A., Smith M. E., Kerr I. B., Bodmer W. F., Gannon J. V., Lane D. P. p53 mutations in colorectal cancer. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7555–7559. doi: 10.1073/pnas.87.19.7555. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rosenberg A. H., Lade B. N., Chui D. S., Lin S. W., Dunn J. J., Studier F. W. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene. 1987;56(1):125–135. doi: 10.1016/0378-1119(87)90165-x. [DOI] [PubMed] [Google Scholar]
- Santhanam U., Ray A., Sehgal P. B. Repression of the interleukin 6 gene promoter by p53 and the retinoblastoma susceptibility gene product. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7605–7609. doi: 10.1073/pnas.88.17.7605. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shaulsky G., Ben-Ze'ev A., Rotter V. Subcellular distribution of the p53 protein during the cell cycle of Balb/c 3T3 cells. Oncogene. 1990 Nov;5(11):1707–1711. [PubMed] [Google Scholar]
- 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]
- Shaulsky G., Goldfinger N., Peled A., Rotter V. Involvement of wild-type p53 in pre-B-cell differentiation in vitro. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):8982–8986. doi: 10.1073/pnas.88.20.8982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shaulsky G., Goldfinger N., Peled A., Rotter V. Involvement of wild-type p53 protein in the cell cycle requires nuclear localization. Cell Growth Differ. 1991 Dec;2(12):661–667. [PubMed] [Google Scholar]
- Shaulsky G., Goldfinger N., Rotter V. Alterations in tumor development in vivo mediated by expression of wild type or mutant p53 proteins. Cancer Res. 1991 Oct 1;51(19):5232–5237. [PubMed] [Google Scholar]
- 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]
- Stürzbecher H. W., Brain R., Maimets T., Addison C., Rudge K., Jenkins J. R. Mouse p53 blocks SV40 DNA replication in vitro and downregulates T antigen DNA helicase activity. Oncogene. 1988 Oct;3(4):405–413. [PubMed] [Google Scholar]
- Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vogelstein B. Cancer. A deadly inheritance. Nature. 1990 Dec 20;348(6303):681–682. doi: 10.1038/348681a0. [DOI] [PubMed] [Google Scholar]
- Wang E. H., Friedman P. N., Prives C. The murine p53 protein blocks replication of SV40 DNA in vitro by inhibiting the initiation functions of SV40 large T antigen. Cell. 1989 May 5;57(3):379–392. doi: 10.1016/0092-8674(89)90913-6. [DOI] [PubMed] [Google Scholar]
- Wilcock D., Lane D. P. Localization of p53, retinoblastoma and host replication proteins at sites of viral replication in herpes-infected cells. Nature. 1991 Jan 31;349(6308):429–431. doi: 10.1038/349429a0. [DOI] [PubMed] [Google Scholar]
- Wolf D., Harris N., Goldfinger N., Rotter V. Isolation of a full-length mouse cDNA clone coding for an immunologically distinct p53 molecule. Mol Cell Biol. 1985 Jan;5(1):127–132. doi: 10.1128/mcb.5.1.127. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wolf D., Rotter V. Major deletions in the gene encoding the p53 tumor antigen cause lack of p53 expression in HL-60 cells. Proc Natl Acad Sci U S A. 1985 Feb;82(3):790–794. doi: 10.1073/pnas.82.3.790. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]