טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentBeno Itai
SubjectThe Molecular Basis of Differential Binding of Wild Type
P53 to its Target Sites
DepartmentDepartment of Biology
Supervisor Professor Tali Haran
Full Thesis textFull thesis text - English Version


Abstract

The tumor-suppressor protein p53 is among the most effective of the cell's natural defenses against cancer, and the DNA binding activity of p53 is pivotal to its tumor suppressor activity. In response to cellular stress, p53 acts as a transcription factor (TF) by binding to defined DNA targets, thereby activating the expression of genes leading to cell-cycle arrest, DNA repair, or apoptosis. Abrogation of p53 sequence-dependent binding is implicated in approximately half of all known cancers. Eighty to 90% of the missense mutations identified in human tumors, are found in the DNA binding domain of p53. This underscores the importance of sequence-specific DNA binding by p53 in maintaining genomic integrity and preventing tumor formation. The consensus sequence of p53 DNA binding sites is highly degenerate; however most validated p53 binding sites deviate from the consensus in at least one position. Despite the vast array of experimental data, the molecular mechanisms by which p53, at different levels, recognize and bind to its DNA targets have remained a puzzle. Better understanding of the complexity of how p53 functions through sequence-specific binding to its DNA targets is a prerequisite for any attempt to restore the function of mutant p53 proteins, or to block their activity, as a potential treatment for cancer. In my experiments I employed the cyclization kinetics method using fluorescence resonance energy transfer to reveal the structural and mechanical properties of the DNA targets in solution, both for free DNA molecules and for bound p53-DNA complexes. Using this method, I determined the magnitude, direction, and mechanical properties of global bending, bending flexibility, torsional flexibility, and helical repeat of p53 target sites. Protein binding microarray (PBM) experiments were performed for parallel high-throughput p53 DNA binding measurements. PBM technology allows identifying in vitro binding selection of a TF by using a dsDNA microarray to determine the rank order of cis-binding sites. An amount of 105k features were designed with designated software to generate a custom microarray. All combinations of eight variable bases in p53 half site were included on the chip by constructing each test sequence (20 bp length) from two identical half-sites (10 bp length). Different p53 proteins were purified and PBM protocols were adjusted to suite p53 binding conditions. Cyclization kinetics method was also used to test the influence of long spacers on p53 conformation and to answer the question of whether p53 imposes an appreciable bend angle on its response elements, with and without spacer sequences up to 20-bp long between half sites. These results of global structural DNA bend and twist parameters conflict with previously published results that suggested, based on evidence from gel-based phasing analysis, that p53 bends its specific target DNA to various extents, from 30° to 60°. My results correlate with crystallographic studies demonstrating that p53DBD did not display any significant bends with various DNA targets.