Is the source of selectivity in p53/DNA interaction resides on the thermodynamic or kinetic aspects of the interaction

The tumor suppressor protein p53 is a transcription factor (TF) that sits at a hub of a complex network in living cell. In response to various types of cellular stress, regulates the expression of a variety of genes involved in DNA damage response, by binding sequence-specifically to defined DNA targets. p53 is mutated in half of all cancers and the vast majority of those mutations are in p53’s DNA binding domain. The sources of selectivity in p53 interactions with its response elements (REs) and its relationship to the sequence-dependent structural code of DNA readout by p53 are currently unknown. On the one hand, the sequence logo of p53 REs is extremely degenerate, where only C4 and G7 in each half sites are information rich. On the other hand, we have previously shown that transactivation is kinetically determined at low levels and thermodynamically driven at high levels. To address the mechanistic origin of selectivity in p53/DNA interactions we have carried out high-throughput selection studies using SELEX-seq, as a function of thermodynamic versus kinetic selection criteria at several different limiting protein concentrations, and have computationally analyzed the DNA binding specificities of p53 to the selected sequences, in the two types of selections. Preliminary results from these experiments demonstrate that the dynamic range of selection by kinetic stability is significantly greater than for thermodynamic affinity, indicating that stability may have a greater effect on the selectivity of p53 to its target REs than DNA binding affinity.