Dynamics of higher order nuclear architecture upon DNA damage

DNA双链断裂（DSB）是电离辐射引起的最严重的细胞损伤，因为不能正确修复它们可能导致肿瘤发生性染色体易位。几种修复蛋白显示在γ辐射后形成更高级的核结构，辐射诱导的修复灶（RIRF）。然而，在人类细胞中，DNA损伤后细胞核结构是如何重组的却知之甚少。为了研究电离辐射后核结构的动态重组，我们研究了RIRF的拓扑和时序关系。为此，我们应用免疫荧光分析研究了4种以上蛋白质在同一细胞核中的定位。利用该技术，我们研究了γ-H2 AX、RAD 51、53 BP 1和泛素化蛋白（FK 2灶）在γ射线照射后不同时间点在人成纤维细胞系中形成的RIRF的定位。结果，我们发现在正常细胞中，在2戈伊的γ射线照射后30分钟，在形成g-H2 AX焦点的核位点处形成FK 2焦点。在2戈伊电离辐射诱导DSB后超过1小时，在γ-H2 AX/FK 2焦点处形成53 BP 1焦点。在12戈伊电离辐射后超过1小时观察到g-H2 AX/FK 253 BP 1灶形成。为了研究ATM和连接酶IV在RIRF形成中的作用，我们对ATM缺陷细胞进行电离辐射。2和12戈伊照射后RIRF的形成显着抑制ATM缺陷细胞表明电离辐射后的RIRF形成的ATM参与。另一方面，连接酶IV缺陷的细胞，携带DSB的末端连接修复系统被干扰，在2和12戈伊照射下均显示出显著更高数量的γ-H2 AX/FK 2灶，表明泛素化系统参与DSB的重组修复途径。

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.