Elimination of radiation-induced gamma-H2AX foci in mammalian nucleus can occur by histone exchange |
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Authors: | Svetlova Maria Solovjeva Liudmila Nishi Kayoko Nazarov Igor Siino Joseph Tomilin Nikolai |
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Affiliation: | Institute of Cytology RAS, St Petersburg, Russia. |
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Abstract: | Double-strand breaks in mammalian DNA lead to rapid phosphorylation of C-terminal serines in histone H2AX (gamma-H2AX) and formation of large nuclear gamma-H2AX foci. After DNA repair these foci disappear, but molecular mechanism of elimination of gamma-H2AX foci remains unclear. H2AX protein can be phosphorylated and dephosphorylated in vitro in the absence of chromatin. Here, we compared global exchange of GFP-H2AX with kinetics of formation and elimination of radiation-induced gamma-H2AX foci. Maximal number of gamma-H2AX foci is observed one hour after irradiation, when approximately 20% of GFP-H2AX is exchanged suggesting that formation of the foci mostly occurs by in situ H2AX phosphorylation. However, slow elimination of gamma-H2AX foci is weakly affected by an inhibitor of protein phosphatases calyculin A which is known as an agent suppressing dephosphorylation of gamma-H2AX. This indicates that elimination of gamma-H2AX foci may be independent of dephosphorylation of H2AX which can occur after its removal from the foci by exchange. |
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Keywords: | DSB, double-strand DNA break GFP, green fluorescent protein FRAP, fluorescence recovery after photobleaching |
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