Book of Abstracts: Albany 2011
June 14-18 2011
©Adenine Press (2010)
Protein-DNA interactions in the p53/DNA system
The tumor suppressor protein p53 is a transcription factor (TF) that, in response to various types of cellular stress, regulates the expression of a variety of genes involved in cell-cycle control, apoptosis, DNA repair, and cell differentiation (1), by first binding sequence-specifically to defined DNA targets (2). Abrogation of p53 sequence-dependent binding is implicated in ~50% of all known cancers (1). p53 molecules consist of four major functional domains (3). The N-terminus contains a transactivation domain; the core domain contains the sequence-specific DNA binding domain (DBD); and the C-terminal domain (CTD) includes a tetramerization domain (TD) and a regulatory domain that contain a separate sequence non-specific DNA binding activity. The core domain of p53 contains 95% of the missense mutations identified in human tumors (4). This highlights the importance of sequence-specific DNA binding by p53 in maintaining genomic integrity and preventing tumor formation. The consensus DNA response element (RE) consists of two decameric half-sites with the general form RRRCWWGYYY (R=A,G;W=A,T;Y=C,T), separated by a variable number of base pairs (2). The WW doublet in the CWWG center of p53 half-sites is uncontacted with the protein in the crystal structure of p53DBD/DNA complexes (5, 6). Nonetheless, this position is highly conserved in p53 binding sites.
We will present our recent study (7) demonstrating that p53DBD bind consensus sequences differing in the CWWG center with different binding affinities and especially binding cooperativity. The binding cooperativity spans five orders of magnitude and is encoded in the structural properties of this region and in particular in the torsional flexibility of the CWWG motif, as determined experimentally by us. The torsionally flexible CATG motif, connected with binding sites related to cell-cycle arrest genes, is bound with high affinity and low cooperativity by p53DBD. The torsionally rigid CAAG and CTAG motifs are bound with lower affinity and high cooperativity. These motifs are abundant in binding sites associated with low affinity apoptosis-related genes. Our results provide a molecular and structural basis to recent findings that DNA binding cooperativity of p53 modulate the decision between cell-cycle arrest and apoptosis (8).
p53 sits at a hub of cellular network controlling many genes, mostly by its function as a TF that binds sequence-specifically to p53 REs. Knowledge of the full spectrum of p53 cellular connectivity is a prerequisite for a true understanding of what can go wrong when p53 function is disrupted. We have determined, using protein-binding microarrays, the binding affinity of p53DBD-TD to a large fraction of all possible combinations of p53 REs, and will present these results.
Department of Biology, Technion, Technion City, Haifa 32000, Israel