The Loss of γH2AX Signal is a Marker of DNA Double Strand Breaks Repair Only at Low Levels of DNA Damage
2006; Taylor & Francis; Volume: 5; Issue: 10 Linguagem: Inglês
10.4161/cc.5.10.2799
ISSN1538-4101
AutoresFanny Bouquet, Catherine Muller, Bernard Salles,
Tópico(s)DNA and Nucleic Acid Chemistry
ResumoAbstractThe induction of DNA double-strand breaks (DSBs) by genotoxic treatment leads to hightoxicity and genetic instability. Various approaches have been undertaken to quantify thenumber of breaks and to follow the kinetic of DSB repair. Recently, the phosphorylation ofthe variant histone H2AX (named γH2AX), quantified by specific immunodetectionapproaches, has provided a valuable and highly sensitive method to monitor DSBs formation.Although it is admitted that the number of γH2AX foci reflected that of DSBs, contradictoryreports leave open the question of a link between the disappearance of γH2AX signal andDSBs repair. We determined γH2AX expression (i) in cells either proficient or not in DSBsrepair capacity, (ii) after exposure to ionizing radiation (IR) or calicheamicin γ1, aradiomimetic compound, (iii) and by three different immunodetection methods, focinumbering, flow cytometry or Western blotting. We showed here that γH2AX loss correlateswith DSB repair activity only at low cytotoxic doses, when less than 100-150 DSBs breaksper genome are produced, independently of the method used. In addition, in DNA repairproficient cells, the early decrease in the number and intensity of γH2AX foci observed after a2 Gy exposure was not associated with a significant change in the global γH2AX level asdetermined by Western blotting or flow cytometry. These results suggest that thedephosphorylation step of γH2AX may be limiting and that the loss of foci is mediated notonly by γH2AX dephosphorylation but also through its redistribution towards the chromatin.
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