Investigating the kinetic parameters of p53 binding to nucleosomes via single molecule total internal reflection fluorescence (smTIRF) microscopy

Horswill, Ian

Undergraduate Honors Thesis

Investigating the kinetic parameters of p53 binding to nucleosomes via single molecule total internal reflection fluorescence (smTIRF) microscopy Public Deposited

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Citeable URL: https://scholar.colorado.edu/concern/undergraduate_honors_theses/t722hb54r

Abstract

P53 is among the most important proteins in the human body. Its influence is far reaching, affecting numerous biological processes including transcription, the cell cycle, and most importantly, tumor development, serving as the body’s predominant tumor suppressor. A key factor in the above-mentioned roles is the ability of p53 to select and bind to DNA, which it does as a tetramer. A number of biochemical studies have explored the binding interactions of p53 to free/naked DNA; however, less is known about how p53 interacts with its DNA binding site within nucleosomes. This project aims to gain a better understanding of the parameters that impact p53 binding to nucleosomes. To investigate this, the binding kinetics of tetrameric p53 on two different nucleosome constructs containing the p53 consensus sequence were quantified at single molecule resolution using Total Internal Fluorescence Reflection (TIRF) microscopy. Results showed that that when the p53 consensus site was more accessible on the edge of the nucleosome near the entry/exit junction, p53 bound similarly to free DNA. However, p53 displayed slightly accelerated kinetics when the consensus site was occluded within the dyad axis of the nucleosome, though still displaying abundant binding. Furthermore, scrambling the p53 binding site on a nucleosome eliminated binding. Lastly, increased dimeric exchanges were observed when tetrameric p53 was bound to nucleosomes compared to DNA, potentially suggesting a change in mechanism for binding to nucleosomes.

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