DNA-binding mechanism of the Escherichia coli Ada O-6-alkylguanine-DNA alkyltransferase

The C-terminal domain of the Escherichia coli Ada protein (Ada-C) aids in t he maintenance of genomic integrity by efficiently repairing pre-mutagenic O-6-alkylguanine lesions in DNA, Structural and thermodynamic studies were carried out to obtain a model of the DNA-binding process, Nuclear magnetic resonance (NMR) studies map the DNA-binding site to helix 5, and a loop reg ion (residues 151-160) which form the recognition helix and the 'wing' of a helix-turn-wing motif, respectively, The NMR date also suggest the absence of a large conformational change in the protein upon binding to DNA, Hence , an O-6-methylguanine (O(6)meG) lesion would be inaccessible to active sit e nucleophile Cys146 if the modified base remained stacked within the DNA d uplex. The experimentally determined DNA-binding face of Ada-C was used in combination with homology modelling, based on the catabolite activator prot ein, and the accepted base-flipping mechanism, to construct a model of how Ada-C binds to DNA in a productive manner. To complement the structural stu dies, thermodynamic data were obtained which demonstrate that binding to un methylated DNA was entropically driven, whilst the demethylation reaction p rovoked an exothermic heat change. Methylation of Cys146 leads to a loss of structural integrity of the DNA-binding subdomain.