High resolution imaging of changes in the structure and spatial organization of chromatin, gamma-H2A.X and the MRN complex within etoposide-induced DNA repair foci.

The focal accumulation of DNA repair factors, including the MRE11/Rad50/NBS1 (MRN) complex and the phosphohistone variant gamma-H2A.X, is a key cytological feature of the DNA damage response (DDR). Although these foci have been extensively studied by light microscopy, there is comparatively little known regarding their ultrastructure. Using correlative light microscopy and electron spectroscopic imaging (LM/ESI) we have characterised the ultrastructure of chromatin and DNA repair foci within the nuclei of normal human fibroblasts in response to DNA double-strand breaks (DSBs). The induction of DNA DSBs by etoposide leads to a global decrease in chromatin density, which is accompanied by the formation of invaginations of the nuclear envelope as revealed by live-cell microscopy. Using LM/ESI and the immunogold localisation of gamma-H2A.X and MRE11 within repair foci, we also observed decondensed 10 nm chromatin fibres within repair foci and the accumulation of large non-chromosomal protein complexes over three hours recovery from etoposide. At 18 h after etoposide treatment, we observed a close juxtapositioning of PML nuclear bodies and late repair foci of gamma-H2A.X, which exhibited a highly organised chromatin arrangement distinct from earlier repair foci. Finally, the dual immunogold labelling of MRE11 with either gamma-H2A.X or NBS1 revealed that gamma-H2A.X and the MRN complex are sub-compartmentalised within repair foci at the sub-micron scale. Together these data provide the first ultrastructural comparison of gamma-H2A.X and MRN DNA repair foci, which are structurally dynamic over time and strikingly similar in organisation.