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GM1492 human diploid skin fibroblasts derived from a patient with Bloom's syndrome (BS), lack detectable p53 mRNA and protein as shown by Northern and Western blotting, and express an increased RecA-like activity. Here we demonstrate that the p53 gene is grossly intact in GM1492 cells according to Southern blotting. DNA sequencing did not reveal any mutations in the promoter region of p53. A highly sensitive RT–PCR produced a p53 cDNA fragment that was shorter than expected. DNA sequence analysis of p53 cDNA showed that exon 6 was missing, explaining the shorter PCR product. Furthermore, sequencing of genomic DNA revealed a base substitution at the nucleotide preceding the AG splice acceptor site of intron 5. The omission of exon 6 creates a frameshift at the junction of exons 5 and 7, and a premature stop codon in exon 7. The aberrant transcript is predicted to encode a truncated p53 protein containing 189 amino acid residues. Moreover, Western blotting demonstrated elevated HsRAD51 protein levels in GM1492 cells. The lack of sufficient levels of wild-type p53 and increased levels of HsRad51 protein may contribute to the elevated RecA-like activity in the GM1492 fibroblasts.

The p53-induced mouse wig-1 gene encodes a Cys2His2-type zinc finger protein of unknown function. The zinc fingers in wig-1 are connected by long (56–75) amino acid linkers. This distribution of zinc finger domains resembles that of the previously described double-stranded (ds)RNA-binding proteins dsRBP-ZFa and JAZ. Ectopically expressed FLAG-tagged mouse wig-1 protein localized to nuclei and in some cells to nucleoli, whereas GFP-tagged mouse wig-1 localized primarily to nucleoli. Electrophoretic mobility shift assay using a recombinant GST–wig-1 fusion protein showed that wig-1 preferentially binds dsRNA rather than single-stranded RNA or dsDNA. A set of deletion/truncation mutants of wig-1 was tested to determine the dsRNA-binding domain(s) or region(s) in wig-1 that is involved in the stabilization of wig-1–dsRNA complexes in vitro. This revealed that the first zinc finger in wig-1 is essential for binding to dsRNA, whereas zinc fingers 2 and 3 are dispensable. wig-1 protein expressed in mammalian cells also showed a high affinity for dsRNA. wig-1 represents the first confirmed p53-induced gene that encodes a dsRNA-binding protein. This suggests that dsRNA binding plays a role in the p53-dependent stress response.

p53 is a transcription factor that binds double-stranded (ds) DNA in a sequence-specific manner. In addition, p53 can bind the ends of single-stranded (ss) DNA. We previously demonstrated that ssDNA oligonucleotides interact with the C-terminal domain of p53 and stimulate binding to internal segments of long ssDNA by the p53 core domain. Here we show that the p53 C-terminal domain can recognize staggered ss ends of dsDNA. We have mapped the binding site for ssDNA ends to residues 361-382 in human p53 using a p53 deletion mutant (p53-delta 30) lacking the 30 C-terminal amino acid residues and a series of 22mer peptides. The binding site for DNA ends coincides with a region previously implicated in regulation of sequence-specific DNA binding by the core domain. The interaction of the C-terminal regulatory domain with the ends of ssDNA or with the protruding ends of dsDNA stimulates both sequence-specific and non-specific DNA binding via the core domain. Electron microscopy demonstrated the simultaneous binding of p53 to dsDNA and a ssDNA end. These results suggest a model in which interaction of the p53 C-terminal tail with DNA ends generated after DNA damage causes activation of sequence-specific p53 DNA binding in vivo and may thus provide a molecular link between DNA damage and p53-mediated growth arrest and apoptosis.

RNA-binding proteins (RBPs) play important roles in the regulation of gene expression through a variety of post-transcriptional mechanisms. The p53-induced RBP Wig-1 (Zmat3) binds RNA through its zinc finger domains and enhances stability of p53 and N-Myc mRNAs and decreases stability of FAS mRNA. To identify novel Wig-1-bound RNAs, we performed RNA-immunoprecipitation followed by high-throughput sequencing (RIP-Seq) in HCT116 and Saos-2 cells. We identified 286 Wig-1-bound mRNAs common between the two cell lines. Sequence analysis revealed that AU-rich elements (AREs) are highly enriched in the 3'UTR of these Wig-1-bound mRNAs. Network enrichment analysis showed that Wig-1 preferentially binds mRNAs involved in cell cycle regulation. Moreover, we identified a 2D Wig-1 binding motif in HIF1A mRNA. Our findings confirm that Wig-1 is an ARE-BP that regulates cell cycle-related processes and provide a novel view of how Wig-1 may bind mRNA through a putative structural motif. We also significantly extend the repertoire of Wig-1 target mRNAs. Since Wig-1 is a transcriptional target of the tumor suppressor p53, these results have implications for our understanding of p53-dependent stress responses and tumor suppression.

The p53 tumor suppressor protein functions as a transcriptional activator that binds specifically to double-stranded DNA (for a review, see reference 15). p53 is normally expressed at low levels in a latent form that is unable to bind specifically to DNA. Under stress conditions, such as DNA damage, hypoxia, and oncogene activation, p53 is activated; it accumulates due to protein stabilization, leading to initiation of the p53-dependent biological response, i.e., cell cycle arrest and apoptosis. The N-terminal region of the p53 protein (residues 1 to 42) contains an acidic transactivation domain (24), and the hydrophobic core domain, corresponding to residues 102 to 292, has sequence-specific DNA binding activity (3, 8, 18, 25). The C-terminal region harbors a tetramerization domain (5, 21), plus a region that regulates specific DNA binding by the core domain (9), and binds single-stranded DNA ends (2). The ability of p53 to activate transcription from promoters containing p53 binding sites is tightly linked to its tumor suppressor function. Most mutant p53 proteins found in human tumors have amino acid substitutions in the core domain, affecting either residues that are involved in direct contacts with DNA, e.g., Arg 248 and Arg 273 (DNA contact mutations), or residues that are important for maintaining the structural integrity of the core domain, e.g., Arg 175 and Arg 249 (structural mutations) (4). As a result, these mutant proteins are partially or completely deficient for specific DNA binding (reviewed in reference 15). Activation of p53 in response to, for instance, DNA damage appears to involve a conformational shift that activates the specific DNA binding. Latent p53 can be activated for specific DNA binding in vitro by various manipulations of its C-terminal domain, including deletion of the last 30 residues, binding of monoclonal antibody PAb421, phosphorylation (9, 10, 11), and acetylation (6), suggesting a role for the C terminus as a negative regulator of p53 activity. This idea is further supported by the findings that short peptides derived from the C-terminal domain of p53 (residues 369 to 383) can activate specific DNA binding (1, 12, 20, 22). According to the allosteric model for regulation of p53 activity (11, 12), a putative interaction between the C-terminal domain and another region in p53 in a p53 tetramer locks the tetramer in a DNA binding-incompetent state. The addition of an excess of C-terminal peptide, as well as binding of antibody PAb421, C-terminal phosphorylation, or acetylation, would displace the C terminus from its binding site in the p53 molecule and thus allow specific DNA binding by the core domains in a tetramer. Our previous observation that a synthetic peptide derived from the p53 C terminus (peptide 46) can restore the transactivation and growth suppression function of mutant p53 proteins in vivo raised the question of the mechanism of peptide 46-mediated p53 activation (20). While the current allosteric model can explain activation of wild-type p53 by the C-terminal peptide, the mechanism behind peptide-mediated reactivation of mutant p53 proteins carrying substitutions of residues that directly contact DNA or residues important for the structural stability of the core domain remains unclear. Understanding the molecular basis of peptide 46-mediated restoration of mutant p53 function is essential for the development of drugs that can reactivate mutant p53 protein in human tumors. Here we demonstrate that the ability of peptide 46 to activate p53 correlates with its ability to bind p53. Peptide 46-interacting regions were identified within both the core and C-terminal domains of p53. We present evidence that the direct binding of the C-terminal polypeptide, added in trans, to the isolated mutant core domains restores their specific DNA binding.