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Nucleic Acids Research, 2000, Vol. 28, No. 19 3710-3718
© 2000 Oxford University Press

DNA-binding mechanism of the Escherichia coli Ada O6-alkylguanine–DNA alkyltransferase

Philip E. Verdemato, James A. Brannigan1, Christian Damblon, Fabio Zuccotto2, Peter C. E. Moody* and Lu-Yun Lian

Department of Biochemistry, University of Leicester, Leicester LE1 7RH, UK, 1Department of Chemistry, University of York, Heslington, York YO10 5DD, UK and 2Centre for Mechanisms in Human Toxicity, University of Leicester, Leicester LE1 9HN, UK

The C-terminal domain of the Escherichia coli Ada protein (Ada-C) aids in the maintenance of genomic integrity by efficiently repairing pre-mutagenic O6-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 region (residues 151–160) which form the recognition helix and the ‘wing’ of a helix–turn–wing motif, respectively. The NMR data also suggest the absence of a large conformational change in the protein upon binding to DNA. Hence, an O6-methylguanine (O6meG) lesion would be inaccessible to active site nucleophile Cys146 if the modified base remained stacked within the DNA duplex. The experimentally determined DNA-binding face of Ada-C was used in combination with homology modelling, based on the catabolite activator protein, 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 studies, thermodynamic data were obtained which demonstrate that binding to unmethylated DNA was entropically driven, whilst the demethylation reaction provoked an exothermic heat change. Methylation of Cys146 leads to a loss of structural integrity of the DNA-binding subdomain.

* To whom correspondence should be addressed. Tel: +44 116 252 3366; Fax: +44 116 252 3473; Email: pcem1@le.ac.uk Present address: Lu-Yun Lian, Department of Biomolecular Sciences, UMIST, Manchester M60 1QD, UK


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