טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentPratik Vyas
SubjectThe Interactions of Wild-Type p53 with its Response
Elements: the Role of Sequence Spacers in p53/DNA
Interactions
DepartmentDepartment of Biology
Supervisor Professor Haran Tali
Full Thesis textFull thesis text - English Version


Abstract

The tumor suppressor protein p53 is a transcription factor that binds sequence-specifically to defined target genes in the genome leading to diverse cellular outcomes such as cell cycle arrest, DNA repair, cell senescence, and apoptosis. The DNA binding sites or response elements (REs) to which p53 binds are made of two 10 base-pair (bp) half-sites with the general form RRRCWWGYYY (R=A,G;W=A,T;Y=C,T), separated by a variable DNA spacer up to 18-bp. When the half-sites are contiguous, one p53 dimer binds to a half-site and another p53 dimer binds to the adjoining half-site, thus forming a functional p53 tetramer, aided by cooperative protein/protein and protein/DNA interactions. However, about 50% of all functionally validated p53 REs contain spacers between the half-sites. It is not known how p53 binds and accommodates itself to REs in which half-sites are separated by long DNA spacers. Moreover, the functional significance of DNA spacers in p53/DNA interactions remains unclear. In this study, using electrophoretic mobility shift assays (EMSA), it is shown that p53 binds to consensus REs containing long spacers in two different modes: fully specific and hemi-specific. In the specific binding mode, both p53 dimers bind specifically to half-sites, whereas in the hemi-specific binding mode, only one p53 dimer is specifically bound to a DNA half-site and the other is bound to the spacer DNA. Nonetheless, both these binding modes have similar binding affinities, and the hemi-specific complex competes better than the specific complex with genomic DNA. Measurement and analysis of binding affinities of p53 to consensus REs having spacers up to 20-bp revealed that the binding affinities were modulated by the rotational positioning of the half-sites. In other words, the binding affinities were higher when the half-sites were either contiguous or rotated nearly in-phase with the DNA helical repeat, and lower when half-sites were out-of-phase and facing nearly opposite directions.  It is demonstrated that p53 is also capable of forming fully-specific and hemi-specific complexes on two natural p53 binding sites, PTEN (Phosphatase and Tensin Homolog) and CHMP4c (Chromatin Modifying Protein 4C). UV-induced site-specific crosslinking studies, substantiate the hemi-specific binding mode. These results lead to the proposition that p53 REs having long spacers (>= 10-bp) consists of two separate specific half-sites. This scenario could lead to the formation of several p53 tetrameric complexes via hemi-specific binding. These findings provides mechanistic insights into p53/DNA recognition and significantly expand and diversify the network of p53 binding sites in the genome. Moreover, it explains the manner in which p53 binds to clusters of more than one canonical binding site, common in many natural REs.