Structural studies of the mouse p53 DNA binding domain in the presence and absence of DNA

• Main Author: Ho, William C
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2006
• Subjects: Biochemistry
• ISBN: 0542799073; 9780542799075

The p53 tumor suppressor plays a crucial role in maintaining the integrity of the genome by triggering cell death in the event of genomic stress. It functions by binding to DNA and regulating transcription of genes crucial to the cell death pathway. P53 interacts with DNA as a tetramer, or a dimer of dimers by virtue of its C-terminal tetramerization domain. The minimal region needed to recognize sequence specific DNA is the DNA binding domain (DBD), corresponding to residues 100-300, which is the most sensitive to tumor derived mutations. Nearly 50% of cancers have mutated p53, with 95% of those mutations occurring in the DBD. The first part of this thesis addresses ways to rescue DBD mutants which are thermodynamically unstable. The 1.55 Å crystal structure of the mouse DBD was solved providing a high resolution structure and a stable crystal lattice for small molecule association studies. A molecule of Tris was observed bound between the S1 and S10 sheets and in vitro studies reveal that Tris binding can potentially contribute to p53DBD stability. The surface of the DBD was also probed for potential binding pockets by soaking with various organic solvents resulting in the location of a single isopropanol binding site. The DBD alone, without the tetramerization domain can bind to DNA cooperatively as a tetramer, although the presence of the tetramerization domain is required for function in vivo. In the second part of this thesis I present the structure of the dimer mouse p53DBD bound to DNA. The 2.3 Å crystal structure shows that the zinc binding H1 helix and the S8-S9 loop mediates dimer-dimer interactions.