Radiation-induced oxidative damage to the DNA-binding domain of the lactose repressor

Abstract : Understanding the cellular effects of radiation-induced oxidation requires the unravelling of key molecular events, particularly damage to proteins with important cellular functions. Escherichia coli lactose operon is a classical model of gene regulation systems. Its function 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 {gamma} 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 probably responsible of this loss of function. Using circular dichroism (CD), fluorescence spectroscopy and a combination of proteolytic cleavage with mass spectrometry, we examine the state of the irradiated headpiece. CD measurements reveal a dose-dependent conformational change involving metastable intermediate states. Fluorescence measurements show a gradual degradation of tyrosines. Mass spectrometry was used to count the number of oxidations in different regions of the headpiece and to narrow down the parts of the sequence bearing oxidised residues. By calculating the relative probabilities of reaction of each amino acid with OH radicals, we can predict the most probable oxidation targets. By confronting the experimental results with the predictions we conclude that Tyr7, Tyr12, Tyr17, Met42 and Tyr47 are the most likely hot spots of oxidation. The loss of repressor function is thus correlated with chemical modifications and conformational changes of the headpiece.
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Nathalie Gillard, Stephane Goffinont, Corinne Buré, Marie Davidkova, Jean-Claude Maurizot, et al.. Radiation-induced oxidative damage to the DNA-binding domain of the lactose repressor. Biochemical Journal, Portland Press, 2007, 403 (3), pp.463-472. ⟨10.1042/BJ20061466⟩. ⟨hal-00478672⟩

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