Dynamics of higher order nuclear architecture upon DNA damage

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DNA double strand breaks (DSBs) are most serious cell damage by ionizing irradiation because failure to properly repair them may lead to tumorigenetic chromosomal translocations. Several repair proteins are shown to form higher order nuclear structures, radiation induced repair foci (RIRF), after gamma irradiation. However, how nuclear structure is reorganized upon DNA damage in human cells is poorly understood. To investigate the dynamic reorganization of nuclear structure after ionizing irradiation, we examined the topological and chronological relationships of RIRF. For this purpose, we applied multicolor immunofluorescence analysis to study the localization of more than 4 proteins in the same nucleus. Using this technique, we studied the localization of RIRF formed by γ-H2AX, RAD51, 53BP1 and ubiquitinated proteins (FK2 foci) in human fibroblast cell lines at various time points after gamma irradiation. As a result, we found that FK2 foci are formed at nuclear sites where g-H2AX foci are formed at 30 min after gamma irradiation at 2 Gy in normal cells. 53BP1 foci are then formed at γ-H2AX/FK2 foci more than one hour after induction of DSBs by 2 Gy ionizing irradiation. The g-H2AX/FK253BP1 foci formation was observed more than one hour after 12 Gy ionizing irradiation. To study the role of ATM and ligase IV in RIRF formation, we performed ionizing irradiation of ATM deficient cells. Formation of RIRF both after 2 and 12 Gy irradiation was significantly repressed in ATM deficient cells suggesting the involvement of ATM in RIRF formation after ionizing irradiation. On the other hand, ligase IV deficient cells, carrying the disturbed end-joining repair system of DSBs, showed significantly higher numbers of γ-H2AX/FK2 foci both at 2 and 12 Gy irradiation suggesting the involvement of ubiquitination system in recombinational repair pathway of DSBs.