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 the 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 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 O:(6)-methylguanine (O:(6)meG) 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.
Verdemato
P E
PE
Department of Biochemistry, University of Leicester, Leicester LE1 7RH, UK.
Brannigan
J A
JA
Damblon
C
C
Zuccotto
F
F
Moody
P C
PC
Lian
L Y
LY
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D002151:Calorimetry; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D019277:Entropy; D004926:Escherichia coli / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017186:Titrimetry
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D004247:DNA; D019853:O(6)-Methylguanine-DNA Methyltransferase