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Title: An investigation into POT1-DNA interactions
Author: Warner, Lyn
Awarding Body: University of Nottingham
Current Institution: University of Nottingham
Date of Award: 2012
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The single stranded DNA binding protein POTI is a member of the Shelterin complex and is known to protect the telomere from DNA damage. It comprises two 0B folding domains responsible for recognising and binding its cognate DNA, as well as a protein binding domain which binds other members of the complex. It is thought that the protein represents a potential target in cancer therapy. The current study aimed to improve our understanding of the validity of POTI as a drug target as follows: 1. Experiments were conducted to examine the effect of manipulating POTI levels on cancer related cellular properties. 2. As prelude to a drug targeting strategy. the contribution of individual bases in the single stranded POTI DNA target region to POTI binding was determined. 3. The cellular effects of several compounds, identified in the laboratory as having POTl-DNA modulating properties, were investigated. In order to acquire a better grasp of the functions of the protein, as well as the roles played by the different domains, cell lines were generated expressing increased levels of POTI or (by dominant negative strategy) decreased levels of POTl. Constructs containing the full length and the DNA binding domains were transfected into HeLa cervical cancer cells leading to expression of the respective coded regions as shown by reverse transcription PCR and Western blots. Associated changes in cell behaviour were studied. When cells were grown in medium supplemented with low levels of foetal bovine serum (0.5%). it was observed that the intrinsic apoptotic pathway, via caspase 9 activation was elevated in the cells expressing the OB domains, leading to a greater overall apoptotic response in these cell lines upon induction. The results suggest a dominant negative effect of OB overexpression, leading to greater sensitivity to apoptosis possibly via destabilisation of the shelterin complex. In addition, the data suggest that overexpression of the full length construct confers a slight protective effect on the cells. To assess the binding between ssDNA and hPOTI-OB, the impact of mutating each residue of the minimal 10mer essential for POTI binding was assessed, using a fluorescence polarisation assay to measure binding. All bases were observed to contribute to binding. yet no mutations led to complete inhibition. The loss of a thymine base at position 7 resulted in a loss of binding albeit moderate. The thymine 7 pyrimidine ring stacks between a tyrosine sandwich site on POTI and the properties of the binding area suggest that this may be a druggabJe binding site. Previous work had identified a set of compounds that based on in silico docking studies and in vitro assessment of inhibition of POTI-DNA binding would bind POTI at this poSition. In the present thesis, four of these compounds which displayed consistent inhibitory activity were further analysed in our cellular assay systems. Compounds caused apoprosis and DNA damage, and inhibited growth. Importantly, the overexpression ofPOTl anenuated apoptotic events for one compound. In conclusion, POTI levels are crucial in the intrinsic apoptotic pathway, possibly due to its stabilising effects. These findings add to mounting evidence that POTI is a promising potential target in cancer therapy. Further to this, compounds that inhibit the interaction between this protein and ssDNA in vitro by disturbing the interaction at T7, show growth inhibition by increasing apoptosis and DNA damage. In one case, this response appears dependent on POT1.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available