|M.Sc Thesis||Department of Biology|
p53 is a tumor suppressor protein that regulates the expression of a variety of genes involved in cell-cycle control, apoptosis, DNA repair and other processes. The protein has a modular structure and can be divided into three main domains: the N-terminal domain, the core or DNA-binding domain (DBD), that binds to specific DNA target sites, and the C-terminal domain that includes tetramerization domain and regulation domain.
In most sequence-specific DNA binding proteins sequence selectivity is based on direct hydrogen bonds between amino acid residues and the donor and acceptor groups (“direct readout”). In addition, sequence-dependent features of DNA structure and flexibility contribute significantly to specific interaction (“indirect readout”).Wild type p53 binds sequence specifically to DNA binding sites consisting of at least two decameric repeats of the general form: RRRCWWGYYY (R=A/G, W=A/T, Y=C/T) mostly as tetramers.
Our working hypothesis is that both direct as well as indirect readout in p53-DNA interactions, conferred by intrinsic conformation and flexibility of the DNA binding sites, plays a central role in modulating this interaction. I used the EMSA technique to determine the thermodynamic parameters of interaction between p53DBD and its binding sites.
I started with three consensus sequences that are identical to one decameric repeat (of two adjacent ones) that was identified as a strong p53-binding site, except for the centeral base pair step which is not directly contacted by p53. The main variability in this series is the stoichiometry of binding ranging from two shifted bands to one. I determined the exact binding stoichiometry of the reaction and found that the stoichiometry of interaction involves preformed dimers of p53 protein, which binds very cooperatively as dimers and tetramers. Then I searched for the differences in thermodynamic parameters when I changed the base pairs known to be responsible for direct readout. This resulted in affinity changes in correlation with the crystal structure of p53DBD to these sites.
Insertion of two base pairs between the two decamer repeats, as known to occur in many natural binding sites, resulted in a binding pattern of two shifted bands, of equal intensity. In addition, this insertion leads to lowering of the binding affinity and stability.
The effect of adding the N-terminus to the DBD was then studied and I found out that the binding affinity of p53TADDBD is significantly lowered compared to its binding to p53DBD alone.