A co-crystal structure of a minimal core p53 DNA binding domain peptide (p53DBD) complexed with a response element and chemical footprinting, cross- linking and cyclization experiments on the complex between wild- type p53 with the WAF1 response element has allowed the construction of a model of the organization of p53 tetramers bound to the recognition site. Our results have shown that, to relieve steric clashes among the four bound peptides, an overall DNA bend of 50| occurs. We have used circular permutation assays to determine the extend and location of the DNA bend. We have found that there is a direct correlation between the DNA bending angle and the binding affinity of the p53DBD with response elements. Our results suggest that the sequence- dependent structural properties of the DNA modulate the stability of p53DBD nucleoprotein complexes and may have profound effects on the function of wild-type and mutated p53. Damage to DNA produced by ionizing radiations, ultraviolet light, and a variety of chemical mutagens induces several cellular responses including the arrest of cell cycle progression, activation of DNA repair, the induction of specific gene products, and in some circumstances, apoptosis. The mechanisms that detect DNA damage and transmit response signals are being characterized and involve p53 and a family of kinases that phosphorylate p53 and other proteins involved in damage response pathways. We have obtained recent data showing that phosphorylation at specific sites is involved both in inducing the accumulation of p53 protein and in activating its site-specific DNA binding ability. Other phosphorylations modulate interactions between p53 and the transcription apparatus, the mdm2 protein, and the DNA repair proteins. Since the radioactive phosphate used to detect phosphorylation itself induces DNA damage, we have developed polyclonal antibodies that specifically detect p53 only when it is phosphorylated at a particular site. These polyclonal antibodies, while extremely useful, are limited in several ways. We are attempting, therefore, to derive monoclonal cell lines that produce a single antibody protein that recognizes a unique epitope. Recently, we have identified a novel transcript whose expression is induced in response to ionizing radiations in a p53-dependent manner, and that shows homology to a type 2C protein phosphatase. Ectopic expression of this new phosphatase in human cells suppressed colony formation. Our results indicate that some of the p53 functions could be initiated by activation of such signaling pathway(s) through the induction of expression of this novel phosphatase.
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