טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentLevitine Michael
SubjectUnravelling the Binding Mechanism of p53 Protein to its
DNA Consensus Sequences
DepartmentDepartment of Biology
Supervisor Professor Tali Haran
Full Thesis textFull thesis text - English Version


Abstract

One of the basic and most important processes, required for the function of cells (for example cell division or start of protein synthesis) is interactions between proteins and DNA. There are two modes of protein::DNA interactions: direct readout that relies on specific hydrogen bonds between protein side chains and the donor and acceptor in DNA major groove; and indirect readout that relies on structural recognition of DNA by proteins. p53 is a tumor suppressor protein that is responsible for genome stability. It acts as a transcription factor and relies on both modes of protein::DNA interactions.
p53 consensus binding site consist of two half sites. The DNA sequence for each half site is R1R2R3C4W5W6G7Y8Y9Y10 (R = A/G; W = A/T; Y = C/T) and p53 has been shown to act as an activator in such sites. The main focus of my research was on bases 5 and 6 - W5W6. It was previously shown in the crystal structure of p53 tetramer bound to its DNA consensus sequence, that only 3 base pairs (bp) of the pentamer (R2R3C4) play a role in forming hydrogen bonds with p53 monomer. The central W5W6 is uncontacted. Nevertheless, most p53 binding sites contain AT or AA step, and not TA steps. I found that the stoichiometry of p53::DNA complexes is dependent on these bases; there are two complexes (tetramer and dimer) for sequences with W5W6 = AT and one complex for sequences with W5W6 = TA or AA. The difference in binding patterns comes from the difference in structural properties of these sequences. The GATC sequence is much more flexible than GTAC or the GAAC sequence. This flexibility ensures that two monomers of p53 are in the right orientation that permits creation of stable bonds between them, therefore stabilizing the dimer.
Furthermore, in my studies I found that base-pairing in W5W6 bases is not obligatory for binding and that binding can occur even without Watson-Crick base pairing, however insertion of even 1 bp spacer between W5 and W6 nullify the binding of p53 to such sequence. Other experiments were performed with different sequences that contain only one half / quarter site. Those experiments proved that the structure of W5W6 is more important than the actual sequence of this site.