Identification des dommages radio-induits de l'ADN et de leurs mécanismes de formation
Jean-Luc Ravanat, Thierry Douki

Induction of damage to DNA is a key event in the lethal and mutagenic effects of ionizing radiation. Extensive studies are carried in the LAN group in order to determine the chemical mechanisms leading to DNA lesions and quantify their formation in cells. In radiobiology, two main pathways are emphasized, namely the direct ionization of DNA and the degradation by the radicals produced upon radiolysis of water (indirect effect).

 

A first step in the elucidation of DNA degradation pathways is to study model systems such as free nucleosides and isolated genomic DNA. Large amount of work was devoted to the chemistry of the guanine radical cation, the major radical generated by direct ionization of DNA. Addition of water to this intermediates leads to the formation of 8-oxo-7,8-dihydroguanine as the major final lesion in double-stranded DNA. Surprisingly, only traces amounts of this modified bases are found at the nucleoside level because it is much more easily oxidized that its precursor and does not accumulate. Nucleophilic addition to the guanine radical cation was also found to be a potential source of crosslink either to proteins via lysine residues or to polyamines.

 

Formation of a complex DNA lesion following attack of a single hydroxyl radical.

Reaction of hydroxyl radicals produced upon radiolysis of water is the other main DNA degradation pathway in cells. Use of HPLC associated to tandem mass spectrometry made possible the identification of new lesions produced in this way. In particular, systematic search for unknown modified bases in the mixture of nucleosides released by enzymatic digestion of irradiated isolated DNA allowed us to identify four new lesions. Among them most attention has been focused on the formation of a complex lesion resulting from the addition of a cytosine base onto the aldehydic residue of an oxidized sugar moiety. This type of damage constitutes a first example of complex DNA damage (more then one lesion in one or two helix turns) that could be generated by a single oxidation event (Fig 1). Evidence was obtained for the formation and repair of this damage in human cells exposed to gamma radiation.

 

The relative contribution of the direct and indirect effects of ionizing radiation to DNA damage is a matter of debates for years. In order to provide new information, we quantified a series of oxidized bases in the DNA of irradiated cells, knowing that ionization of DNA leads to predominant guanine damage while action of hydroxyl radicals is much less specific. These analyses were carried out upon exposure not only to gamma rays but also to heavy ions (GANIL Caen) that are expected to favor direct ionization. A profile consistent with the chemistry of the hydroxyl radical was observed with all radiations. Moreover, the overall yield of oxidized bases decreased when the linear energy transfer of the particle increased, in close correlation to the radiolytic yield of hydroxyl radicals. Direct ionization seems thus to be a minor process in cells.

 

Selected publication(s)  S. Perrier et al.,  J. Am. Chem. Soc. 128 (2006) 5703.  P. Regulus et al.  Proc. Natl. Acad. Sci. USA 104, (2007) 14032. T. Douki et al.,. Int. J. Radiat. Biol. 82 (2006) 119.

 

Maj : 08/09/2014 (1066)

 

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