Repair of Alkylated DNA by the E. coli Ada Protein

Verdemato, P. E.; Moody, P.C.E.

doi:10.1007/978-3-642-48770-5_1

Cited by 1 publication

(6 citation statements)

References 117 publications

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“…Backbone assignment of wild-type Ada-C was performed using a 1.3 mM sample of 70% deuterated, 15 N/ 13 C-labelled Ada-C in 50 mM sodium phosphate buffer pH 6.7 (90% H 2 O, 10% 2 H 2 O), 10 mM 2 H-DTT, 1 mM 2 H-EDTA, 100 mM NaCl and 100mM NaBr. Each triple resonance experiment was carried out in the gradient-enhanced, sensitivity-enhanced mode.…”

Section: Nmr Assignment Of the Ada-c Backbonementioning

confidence: 99%

“…15 N-labelled, 70% deuterated inactive C146S Ada-C was also complexed with both double-and single-stranded methylated and unmethylated DNA for a comparison of binding modes.…”

Section: Dna-binding Analysis By Nmrmentioning

confidence: 99%

“…Demethylation reactions were also carried out in situ in the NMR tube by the addition of ssDNA containing an O 6 meG to 15 N-labelled, 70% deuterated wild-type Ada-C in various ratios. The effect on the Ada-C structure of methyl group transfer to the active site cysteine was then examined by NMR.…”

Section: Dna-binding Analysis By Nmrmentioning

confidence: 99%

“…The tautomeric state of the imidazole sidechain of His147 was determined using 1 H- 15 N HMQC experiments in the manner of Damblon et al (26) for both free and DNA-bound wild-type Ada-C.…”

Section: Dna-binding Analysis By Nmrmentioning

confidence: 99%

“…The function of Ada as a transcription factor is thus dependent on the methylation of Cys69, and the presence of the C-terminal domain (regardless of methylation state) (8,13). This process of transcriptional enhancement is known as the 'adaptive response' and leads to further production of Ada protein capable of repairing alkylated DNA (14,15).…”

Section: Introductionmentioning

confidence: 99%

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

Verdemato¹

2000

Nucleic Acids Research

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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.

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Section: Nmr Assignment Of the Ada-c Backbonementioning

confidence: 99%

“…15 N-labelled, 70% deuterated inactive C146S Ada-C was also complexed with both double-and single-stranded methylated and unmethylated DNA for a comparison of binding modes.…”

Section: Dna-binding Analysis By Nmrmentioning

confidence: 99%

Section: Dna-binding Analysis By Nmrmentioning

confidence: 99%

“…The tautomeric state of the imidazole sidechain of His147 was determined using 1 H- 15 N HMQC experiments in the manner of Damblon et al (26) for both free and DNA-bound wild-type Ada-C.…”

Section: Dna-binding Analysis By Nmrmentioning

confidence: 99%