|M.Sc Student||Jessy Safieh|
|Subject||Is the Functional Outcome of p53 Dependent Genes Encoded in|
the Flexibility of p53 Response Elements?
|Department||Department of Biology||Supervisor||Professor Haran Tali|
|Full Thesis text|
Sequence specific recognition of DNA by regulatory proteins relies on the interaction of sequence-specific proteins with specific DNA functional groups, along with indirect effects that reflect variable energetics in the response of DNA sequences to twisting and bending distortion induced by the protein. Sequence-dependent DNA structure and flexibility are important features of the DNA helix, because they influences almost every aspect of DNA-related processes. The idea that DNA sequence information might also be encoded through its three-dimensional shape and mechanical properties provide a new aspect of thinking about functionality, helping us understand the relative binding affinity of proteins as a function of the structure and mechanical properties of their response elements (REs) on the DNA.
p53 acts as a transcriptional factor by binding to DNA targets, leading to the expression of many genes that participate in a variety of biological processes in response to stress, including cell-cycle arrest, DNA repair, and apoptosis. The consensus sequence of p53 RE is highly degenerate and consists of two decameric half-sites with the motif RRRCWWGYYY (R= A,G; W= A,T; Y= C,T), separated by a variable DNA spacer up to 18 base-pairs. Abrogation of p53 sequence-dependent binding is implicated in ~50% of all known cancers.
The aim of this study was to understand the relationship between the flexibility of p53 REs and functional selectivity of p53. According to our hypothesis, based on calculations of the average deformability of all validated p53 REs with zero spacer grouped by their functional outcome, we suggest to divide p53 REs into two groups. The first group has low deformability value and includes genes that promote permanent or temporary cell-cycle arrest, that we suggest to be called "early" or "basal", since the genes in this group are suggested to be transactivated under low levels of p53. The second group has low deformability values, and includes genes that promote an apoptotic response, that we suggest to be called "late" or "induced" since the genes in it are suggested to be transactivated at higher levels of p53. I validated this hypothesis by experimentally measuring the flexibility of six validated natural p53 REs full sites, with zero base-pair spacer, of which two are related to DNA repair, two related to cell cycle, and two related to apoptosis functions. The measurements were done by the cyclization kinetics assay using fluorescence resonance energy transfer (FRET). The cyclization kinetics assay allows the quantitation of global aspects of DNA structure and dynamics, by measuring the probability of helix formation between two cohesive ends of a linear DNA. In addition, I used electrophoretic mobility shift assay (EMSA) to determine binding affinity and cooperativity of p53 to the R2 p53 target site. I showed that natural p53 REs are relatively straight, and are 2 similar to each other with regard to their bending flexibility. In contrast, the studied p53 REs display a wide range of torsional flexibility values. Moreover, the analyses showed that repair related REs as a group are significantly more flexible than apoptosis related REs.