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Sequence-specific DNA binding by p53: identification of target sites and lack of binding to p53 - MDM2 complexes
A Zauberman1, Y Barak, N Ragimov
1Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.
Abstract:
An immune selection procedure was employed in order to isolate p53 binding sites from mouse genomic DNA. Two DNA clones capable of tight specific interaction with wild type p53 were subjected to further characterization. In both cases, the p53 binding regions displayed a high degree of sequence homology with the consensus binding site defined for human genomic DNA. One of the clones was found to be derived from the LTR of a retrovirus-like element (a member of the GLN family). The region encompassing the GLN LTR p53 binding site could confer p53 responsiveness upon a heterologous promoter. Furthermore, the expression of the endogenous, chromosomally integrated GLN elements was significantly induced upon activation of wild type p53 in cells harboring a temperature sensitive p53 mutant. Finally, it was demonstrated that p53 - MDM2 complexes fail to bind tightly to such a p53 binding site. This may contribute to the inhibition by MDM2 of p53-mediated transcriptional activation.
Insights
Researchers identified specific DNA binding sites for the p53 protein in mouse DNA. These sites, found in a retroviral element, are responsive to p53 and are not bound by p53-MDM2 complexes.
Area of Science:
- Molecular Biology
- Genomics
- Cancer Research
Background:
- The p53 protein is a crucial tumor suppressor involved in DNA repair and apoptosis.
- Identifying specific p53 binding sites is essential for understanding its transcriptional regulation.
- Mouse genomic DNA serves as a model for studying gene regulation.
Purpose of the Study:
- To isolate and characterize p53 binding sites from mouse genomic DNA.
- To investigate the functional significance of identified p53 binding sites.
- To explore the interaction of p53-MDM2 complexes with these sites.
Main Methods:
- Immune selection to isolate p53 DNA binding clones.
- DNA sequencing to determine homology with known p53 binding sites.
- Reporter gene assays to assess promoter responsiveness.
- Analysis of gene expression in cells with temperature-sensitive p53 mutants.
Main Results:
- Two DNA clones with high affinity for wild-type p53 were isolated.
- One clone contained a p53 binding site within a GLN family retroviral LTR.
- This GLN LTR conferred p53 responsiveness and was induced by p53 activation.
- p53-MDM2 complexes showed reduced binding to this p53 site.
Conclusions:
- Mouse genomic DNA contains functional p53 binding sites with homology to human consensus sites.
- Retroviral LTRs can harbor p53 responsive elements.
- The identified p53 binding site may evade MDM2-mediated inhibition, impacting p53 transcriptional activity.