University of Leicester
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The interaction of the Ada protein with DNA : structure and thermodynamics

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posted on 2014-12-15, 10:32 authored by Philip Edward. Verdemato
The C-terminal domain of the E. coli Ada protein (Ada-C) aids the maintenance of genomic integrity by efficiently repairing pre-mutagenic O6-alkylguanine lesions in DNA. The aim of this thesis was to discern the manner in which Ada-C binds and repairs DNA. The research was pursued from both structural and thermodynamic perspectives, to obtain a model of the DNA-binding process.;The production of recombinant Ada-C from E. coli culture was enhanced through a combination of rational media design, E. coli strain choice, and the employment of a growth strategy for maximising cell density prior to induction of protein expression.;Nuclear Magnetic Resonance (NMR) studies mapped the DNA-binding site to the recognition helix of the helix-turn-helix motif and a loop region (residues 149-155) known as the 'wing'. Using this binding interface, and in the absence of a large conformational change in the protein upon DNA-binding, it was found that an O6 meG lesion was inaccessible to active site nucleophile Cys 144 when the lesion remained stacked within the DNA duplex. This lesion could enter the active site by being rendered extrahelical, or "flipping".;The DNA-binding process was shown to be entropically driven, whilst the demethylation reaction provoked an exothermic heat change. At millimolar concentrations, methylation of Ada-C led to a loss of structural integrity.;Duplex DNA containing an O6meG lesion had a lower enthalpy of melting than unmethylated DNA. This, along with 19F NMR work on modified DNA, led to the postulation that O6meG might induce localised duplex melting. This would certainly aid the base-flipping process.


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Awarding institution

University of Leicester

Qualification level

  • Doctoral

Qualification name

  • PhD



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