טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentShraibman Jenia
SubjectThe Kinetics of p53/DNA Interactions
DepartmentDepartment of Biology
Supervisor Professor Haran Tali
Full Thesis textFull thesis text - English Version


Abstract

p53 is a transcription factor that belongs to the family of tumor suppressor genes. In response to various types of cellular stress, it regulates the expression of a variety of genes involved in cell-cycle arrest, apoptosis, DNA repair and more. p53 binds in a sequence-dependent manner to defined DNA targets. Its consensus binding site consists of two half sites, with the general form RRRCWWGYYY (R=A, G; W=A, T; Y=C, T), with a spacer of 0-13 bases between the half sites. Most p53 natural targets differ from the consensus in at least one position. Its functional unit is a tetramer, assembled from two dimers.

p53 binds with high affinity to sequences involved in cell cycle arrest and DNA repair. On the other hand, it binds with various affinities to sequences involved in apoptosis. Its transactivation capacity depends on the protein level and on the specific response element (RE) sequence. I found a correlation between its binding affinity to various DNA targets (Kd) and transactivation at high protein concentration. Such correlation was not found by me when I studied transactivation at basal protein concentration. This means that at basal protein concentration thermodynamic affinity is not the cause for differential transactivation from different targets. Since Kd is the ratio between the dissociation (Koff) constant and association constant (Kon), this ratio may mask the differential effect each of the rate constants may have on kinetics of p53/DNA interactions. For that reason I measured the dissociation constant (Koff) of p53/DNA complexes, to assess whether kinetic stability is at the source of the different transactivation capacities of p53 from different REs at basal conditions. I studied this question by using the Electrophoretic Mobility Shift Assay (EMSA). I found a trend between the kinetic stability of p53DBDtetramer:DNA complexes and transactivation at basal protein levels. I did not find a similar trend between p53DBDdimer:DNA complexes and transactivation at basal protein concentration. This is in the line with the observation that a tetrameric complex is needed for efficient transactivation. I have also measured the kinetic stability of p53DBD:DNA complexes with spacer sequences between half-sites. I observed that a spacer between half-sites significantly decreases the kinetic stability of p53DBDtetramer:DNA complexes.