Abstract
High resolution chemical footprinting and cross-linking experiments have provided a basis for elucidating the overall architecture of the complex between the core DNA binding domain of p53 (p53DBD, amino acids 98-309) and the p21/waf1/cip1 DNA response element implicated in the G1/S phase cell cycle checkpoint. These studies complement both a crystal structure and earlier biophysical studies and provide the first direct experimental evidence that four subunits of p53DBD bind to the response element in a regular staggered array having pseudodyad symmetry. The invariant guanosines in the highly conserved C(A/T)|(T/A)G parts of the consensus half-sites are critical to the p53DBD-DNA binding. Molecular modeling of the complex using the observed peptide-DNA contacts shows that when four subunits of p53DBD bind the response element, the DNA has to bend approximately 50 degrees to relieve steric clashes among different subunits, consistent with recent DNA cyclization studies. The overall lateral arrangement of the four p53 subunits with respect to the DNA loop comprises a novel nucleoprotein assembly that has not been reported previously in other complexes. We suggest that this kind of nucleoprotein superstructure may be important for p53 binding to response elements packed in chromatin and for subsequent transactivation of p53-mediated genes.
Highlights
Wild type p53 is a widely distributed phosphoprotein that has become fundamental in cancer research [1,2,3]
The two TG base elements are separated by 10 bp and are spatially in phase along the helix. p53DBD binding shifts the cleavage frequency minimum by one base toward the 3Ј-end and further diminishes the cleavage frequency at ATGT and TGTT sequences, while the central CCCAAC bases show a relative increase in the cleavage frequency (Fig. 2A, lane F; Fig. 2E, plot b)
These bases are in phase along the helix with a separation of 10 bp and are located in the major groove of the DNA. (iii) Most of the observed contact points are clustered in the major groove with relatively few contacts in the minor groove in agreement with the bound p53DBD in the crystal structure [18]. (iv) The GTTG sequence of the bottom strand shows higher protection from hydroxyl radical cleavage than does the complementary CAAC sequence on the opposite strand, suggesting that it is shielded by bound peptide from the minor groove side, whereas the complementary CAAC sequence is exposed
Summary
Wild type p53 is a widely distributed phosphoprotein that has become fundamental in cancer research [1,2,3]. P53 DNA Binding Domain Nucleoprotein Complexes tural model for the complex of four human p53DBD peptides with an important functional response element: the p21/waf1/ cip binding site This model goes considerably beyond that provided by the earlier crystallographic study and is able to rationalize a number of earlier observations including the requirement for DNA bending in the full tetrapeptide complex. It provides unique insights into possible roles of DNA flexibility in the sequence specificity of p53 binding and suggests possible relationships between the relative orientation of a tetrameric p53 complex on response element DNA and p53 transactivational function
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