Understanding the cellular effects of radiation-induced oxidation requires the unravelling of key molecular events, particularly damage to proteins with important cellular functions. The Escherichia coli lactose operon is a classical model of gene regulation systems. Its functional mechanism involves the specific binding of a protein, the repressor, to a specific DNA sequence, the operator. We have shown previously that upon irradiation with γ-rays in solution, the repressor loses its ability to bind the operator. Water radiolysis generates hydroxyl radicals (OH· radicals) which attack the protein. Damage of the repressor DNA-binding domain, called the headpiece, is most likely to be responsible of this loss of function. Using CD, fluorescence spectroscopy and a combination of proteolytic cleavage with MS, we have examined the state of the irradiated headpiece. CD measurements revealed a dose-dependent conformational change involving metastable intermediate states. Fluorescence measurements showed a gradual degradation of tyrosine residues. MS was used to count the number of oxidations in different regions of the headpiece and to narrow down the parts of the sequence bearing oxidized residues. By calculating the relative probabilities of reaction of each amino acid with OH· radicals, we can predict the most probable oxidation targets. By comparing the experimental results with the predictions we conclude that Tyr7, Tyr12, Tyr17, Met42 and Tyr47 are the most likely hotspots of oxidation. The loss of repressor function is thus correlated with chemical modifications and conformational changes of the headpiece.
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Research Article|
April 12 2007
Radiation-induced oxidative damage to the DNA-binding domain of the lactose repressor
Nathalie Gillard;
Nathalie Gillard
*Centre de Biophysique Moléculaire, CNRS, rue C. Sadron, 45071 Orléans Cedex 2, France
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Stephane Goffinont;
Stephane Goffinont
*Centre de Biophysique Moléculaire, CNRS, rue C. Sadron, 45071 Orléans Cedex 2, France
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Corinne Buré;
Corinne Buré
*Centre de Biophysique Moléculaire, CNRS, rue C. Sadron, 45071 Orléans Cedex 2, France
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Marie Davidkova;
Marie Davidkova
†Nuclear Physics Institute, Department of Radiation Dosimetry, Na Truhlarce 39/64, CZ-18086, Praha 8, Czech Republic
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Jean-Claude Maurizot;
Jean-Claude Maurizot
*Centre de Biophysique Moléculaire, CNRS, rue C. Sadron, 45071 Orléans Cedex 2, France
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Martine Cadene;
Martine Cadene
*Centre de Biophysique Moléculaire, CNRS, rue C. Sadron, 45071 Orléans Cedex 2, France
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Melanie Spotheim-Maurizot
Melanie Spotheim-Maurizot
1
*Centre de Biophysique Moléculaire, CNRS, rue C. Sadron, 45071 Orléans Cedex 2, France
1To whom correspondence should be addressed (email spotheim@cnrs-orleans.fr).
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Publisher: Portland Press Ltd
Received:
September 26 2006
Revision Received:
January 24 2007
Accepted:
January 31 2007
Accepted Manuscript online:
January 31 2007
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2007
Biochem J (2007) 403 (3): 463–472.
Article history
Received:
September 26 2006
Revision Received:
January 24 2007
Accepted:
January 31 2007
Accepted Manuscript online:
January 31 2007
Citation
Nathalie Gillard, Stephane Goffinont, Corinne Buré, Marie Davidkova, Jean-Claude Maurizot, Martine Cadene, Melanie Spotheim-Maurizot; Radiation-induced oxidative damage to the DNA-binding domain of the lactose repressor. Biochem J 1 May 2007; 403 (3): 463–472. doi: https://doi.org/10.1042/BJ20061466
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