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

Philip E. Verdemato; James A. Brannigan; Christian Damblon; Fabio Zuccotto; Peter C. E. Moody; Lu Y. Lian

doi:10.1093/nar/28.19.3710

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

Philip E. Verdemato
, James A. Brannigan
, Christian Damblon
, Fabio Zuccotto
, Peter C. E. Moody (Lead / Corresponding author)
, Lu Y. Lian

Drug Discovery Unit

Research output: Contribution to journal › Article › peer-review

16 Citations (Scopus)

Abstract

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 thermo-dynamic 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 Cys 146 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 Cys 146 leads to a loss of structural integrity of the DNA-binding subdomain.

Original language	English
Pages (from-to)	3710-3718
Number of pages	9
Journal	Nucleic Acids Research
Volume	28
Issue number	19
DOIs	https://doi.org/10.1093/nar/28.19.3710
Publication status	Published - 1 Oct 2000

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

SDG 3 Good Health and Well-being

ASJC Scopus subject areas

Genetics

Access to Document

10.1093/nar/28.19.3710

Cite this

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title = "DNA-binding mechanism of the Escherichia coli Ada O6-alkylguanine-DNA alkyltransferase",

abstract = "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 thermo-dynamic 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 Cys 146 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 Cys 146 leads to a loss of structural integrity of the DNA-binding subdomain.",

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AU - Verdemato, Philip E.

AU - Brannigan, James A.

AU - Damblon, Christian

AU - Zuccotto, Fabio

AU - Moody, Peter C. E.

AU - Lian, Lu Y.

PY - 2000/10/1

Y1 - 2000/10/1

N2 - 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 thermo-dynamic 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 Cys 146 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 Cys 146 leads to a loss of structural integrity of the DNA-binding subdomain.

AB - 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 thermo-dynamic 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 Cys 146 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 Cys 146 leads to a loss of structural integrity of the DNA-binding subdomain.

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