טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentAnna Zivan
SubjectThe Impact of DNA structure and Flexibility of p53 Response
Elements with Long Spacers on p53 Binding Affinity
and Cooperativity
DepartmentDepartment of Biology
Supervisor Professor Haran Tali
Full Thesis textFull thesis text - English Version


Abstract

The tumor suppressor protein, p53, nicknamed “Guardian of the genome”, is a sequence-specific transcription factor involved in many cellular functions. p53 is activated following stress, and as a result is known to trigger several different reactions: a non-exhaustive list of p53-dependant activities includes regulation of DNA metabolism, apoptosis, cell-cycle regulation, senescence, and angiogenesis. Yet, how p53 distinguishes between each type of response is still unknown. The p53 protein spans 393 amino acids, and has three functional domains: for signaling and interaction with other proteins (residues 1 - 93), for sequence-specific recognition of DNA targets (residues 94 - 291), and for tetramerization and additional protein-protein interactions (residues 292 - 393). p53 DNA targets are termed REs (response elements). The REs can be written as a general consensus of two decameric (10 bp) half-sites separated by a random DNA spacer of 0-18 bp (RRRCWWGYYY-spacer-RRRCWWGYYY, where R = A,G; W = A,T; Y = C,T). Previously, it was discovered that DNA structural properties, namely flexibility, influence p53 binding to different REs. This work focuses on the question of how the structural properties of REs with spacers, influence p53/DNA interactions. I propose here that DNA flexural properties affect p53-DNA interactions of p53 REs with spacers. I used quantitative EMSA to systematically evaluate p53 binding affinity to different consensus-like REs constructs with spacers and to natural p53 REs. The advantage of using quantitative EMSA is in its rapidness, robustness, sensitivity, and its ability to distinguish between different bound species. It is shown here that there is a variation in flexibility between natural p53 REs, as a function of the length of spacer between the two REs half-sites. Experimentally, it is shown that without a spacer, p53 shows a preference to bind flexible REs. The addition of flexible spacers does not change p53 preference to flexible REs, but once a rigid spacer is added, p53 has higher binding affinity to mixed flexibility REs. Verification of p53 binding affinity pattern with natural REs showed compatible pattern to that of the consensus-like sequences. To show that the proposed p53 DNA binding pattern is maintained in vivo, select REs were cloned upstream of a luciferase protein to evaluate transactivation levels dependent on p53 presence. Such an assay is prevalent in assessing transcription factors effectiveness and is simple to preform and evaluate. Only initial results were obtained from the in vivo system. This work demonstrates that p53 REs spacer flexibility properties influence p53-DNA binding affinity.