|M.Sc Student||Tucker Scott|
|Subject||High-Throughput Investigation of the Mechanistic|
Origin of p53/DNA Interactions
|Department||Department of Biology||Supervisor||Professor Tali Haran|
|Full Thesis text|
p53 performs a wide variety of cellular functions by activating genes involved in cell-cycle arrest, apoptosis, senescence, DNA repair, and interactions with tumor microenvironments. p53 binds to the highly degenerate consensus site RRRCWWGYYYRRRCWWGYYY, with a spacer region of up to 18 bp between each decametric half-site. p53 binding is dependent on a number of factors including cell or tissue type, active concentration in the nucleus, and posttranslational modifications. p53 is mutated in half of all cancers and the vast majority of those mutations are in p53’s DNA binding domain (DBD). This emphasizes the importance of sequence dependent binding of p53 to its response elements (REs) and highlights the need to understand the mechanism behind p53 binding, which currently remains unsolved, rendering comprehension of p53 binding and subsequent cellular activity difficult.
The aim of this work was to establish the SELEX-seq technique into our laboratory, and use it to study the mechanistic origin of p53/DNA interactions. Specifically, I was interested in studying p53 binding under thermodynamic conditions at various concentrations to understand p53/DNA interactions at a wide range of affinities. Moreover, I examined the dynamics of p53/DNA interactions, that is the kinetic stability of p53 on its REs at various stringencies, to discover the range of stabilities possible for p53/DNA complexes. I used this data to investigate the sources of selectivity in protein-DNA interactions in the p53 system. I expect that a combination of these factors will improve our ability to understand p53 binding activity as the basis of target gene selectivity by p53.
I succeeded in implementing SELEX-seq into our laboratory, and improved it by visualizing the p53/DNA complex with a fluorescent dye rather than approximating its location via a radioactively labeled control DNA. 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 RE. In addition, our data indicates that the central CATG motif may be more important to the stability of p53/DNA complexes than their affinity.