Mladenov Emil, Anachkova Boyka, Tsaneva Irina
Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria.
Genes Cells. 2006 May;11(5):513-24. doi: 10.1111/j.1365-2443.2006.00958.x.
The repair of DNA double-strand breaks involves the accumulation of key homologous recombination proteins in nuclear foci at the sites of repair. The organization of these foci in relation to non-chromatin nuclear structures is poorly understood. To address this question, we examined the distribution of several recombination proteins in subcellular fractions following treatment of HeLa cells with ionizing radiation and the crosslinking agent mitomycin C. The results showed association of Rad51, Rad54, BRCA1 and BRCA2, but not Rad51C, with the nuclear matrix fraction in response to double-strand breaks induction. The association of Rad51 with the nuclear matrix correlates with the formation of Rad51 nuclear foci as a result of DNA damage. Fractionation in situ confirmed that Rad51 foci remained firmly immobilized within the chromatin-depleted nuclei. Irs1SF cells that are unable to form Rad51 damage-induced nuclear foci did not show accumulation of Rad51 in the nuclear matrix. Similarly, no accumulation of Rad51 in the nuclear matrix could be observed after treatment of HeLa cells with the kinase inhibitor caffeine, which reduces formation of Rad51 foci. The results were compared to the distribution of the phosphorylated histone variant, gamma-H2AX. These data suggest a dynamic association and tethering of recombination proteins and surrounding chromatin regions to the nuclear matrix.
DNA双链断裂的修复涉及关键同源重组蛋白在修复位点的核灶中积累。这些核灶与非染色质核结构的关系尚不清楚。为了解决这个问题，我们在用电离辐射和交联剂丝裂霉素C处理HeLa细胞后，检测了亚细胞组分中几种重组蛋白的分布。结果显示，响应双链断裂诱导，Rad51、Rad54、BRCA1和BRCA2与核基质组分相关联，但Rad51C没有。Rad51与核基质的关联与DNA损伤导致的Rad51核灶形成相关。原位分级分离证实，Rad51核灶在无染色质的细胞核内保持牢固固定。无法形成Rad51损伤诱导核灶的Irs1SF细胞在核基质中未显示Rad51积累。同样，在用激酶抑制剂咖啡因处理HeLa细胞后，未观察到Rad51在核基质中的积累，咖啡因会减少Rad51核灶的形成。将这些结果与磷酸化组蛋白变体γ-H2AX的分布进行了比较。这些数据表明重组蛋白和周围染色质区域与核基质之间存在动态关联和连接。
