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Title: Mechanistic and structural studies of the helical arch of flap endonuclease
Author: Patel, Nikesh
ISNI:       0000 0004 2731 7868
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2012
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Fluorescence anisotropy measurements were also carried out in order to determine the dissociation constant of the synthesised oligos bound to FEN enzymes and validate conditions used in the aforementioned competition experiments. These experiments revealed a small dependence on substrate flap length to binding, and showed stimulation of KD on the addition of divalent metal ions. This was likely due to shielding of 7/8 conserved carboxylates that ligand divalent metal ions within the active site of the enzyme. Measurements with hFEN1 mutants, also highlighted the fact that initial binding of substrates is not affected by the helical arch of FEN enzymes. Mutagenesis studies were also carried out. Leucine residues were mutated to proline along the helical arch of human FEN, to disrupt proper alpha helical structure and any disorder-to-order transitions. This gave an extremely deficient phenotype with rates extremely low compared to the wild type enzyme, showing a large significance of the helical arch structure on the activity of the enzyme.
Supervisor: Grasby, Jane Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available