A novel and simple micro-irradiation technique for creating localized DNA double-strand breaks.
A novel and simple micro-irradiation technique for creating localized DNA double-strand breaks.
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DOI：
10.1093/nar/gkq226
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发表时间：
2010-07
影响因子：
14.9
通讯作者：
Yamashita S
中科院分区：
文献类型：
--
作者：
Suzuki K;Yamauchi M;Oka Y;Suzuki M;Yamashita S
共济失调毛细血管扩张突变 (ATM) 依赖性 DNA 损伤信号通过各种因素的相互作用而被放大，这些信号被招募到具有 DNA 双链断裂的染色质区域。通过荧光显微镜结合激光微照射来分析这些因素的空间和时间调节。在这里，我们描述了一种不需要激光源的新颖而简单的微照射技术。细胞用 BrdU 标记 48-72 小时，用多孔聚碳酸酯膜覆盖，并暴露于 UVC。所有 BrdU 标记的细胞在照射后均显示出磷酸化 ATM、磷酸化组蛋白 H2AX、MDC1 和 53BP1 的局部病灶，表明这些病灶的诱导与细胞周期阶段无关。在用 BrdU 标记的核苷酸切除修复缺陷型 XPA 细胞中也可以检测到它们，表明病灶不反映切除修复相关过程。此外，ATM特异性抑制剂显着减弱了病灶形成，并且在非同源末端连接缺陷细胞中显着消除了病灶消失。因此，可以得出结论，微辐射在 BrdU 致敏细胞中产生了 DNA 双链断裂。目前的技术应该加速DNA损伤反应、DNA修复和DNA重组领域的研究，因为它提供了更多的机会在没有任何特定设备的情况下进行微辐照实验。
An ataxia-telangiectasia mutated (ATM)-dependent DNA damage signal is amplified through the interaction of various factors, which are recruited to the chromatin regions with DNA double-strand breaks. Spatial and temporal regulation of such factors is analysed by fluorescence microscopy in combination with laser micro-irradiation. Here we describe a novel and simple technique for micro-irradiation that does not require a laser source. Cells were labelled with BrdU for 48–72 h, covered with porous polycarbonate membranes, and exposed to UVC. All BrdU-labelled cells showed localized foci of phosphorylated ATM, phosphorylated histone H2AX, MDC1 and 53BP1 upon irradiation, showing that these foci were induced irrespective of the cell-cycle phase. They were also detectable in nucleotide excision repair-defective XPA cells labelled with BrdU, indicating that the foci did not reflect an excision repair-related process. Furthermore, an ATM-specific inhibitor significantly attenuated the foci formation, and disappearance of the foci was significantly abrogated in non-homologous end-joining-defective cells. Thus, it can be concluded that micro-irradiation generated DNA double-strand breaks in BrdU-sensitized cells. The present technique should accelerate research in the fields of DNA damage response, DNA repair and DNA recombination, as it provides more chances to perform micro-irradiation experiments without any specific equipment.
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DOI：
10.1083/jcb.200510130
发表时间：
2006-04-24
期刊：
The Journal of cell biology
影响因子：
--
作者：
Bekker-Jensen S;Lukas C;Kitagawa R;Melander F;Kastan MB;Bartek J;Lukas J
通讯作者：
Lukas J
影响因子：
4.8
作者：
Kim, JS;Krasieva, TB;Yokomori, K
通讯作者：
Yokomori, K
影响因子：
64.8
作者：
Friedberg, EC
通讯作者：
Friedberg, EC
DOI：
10.1073/pnas.0810987106
发表时间：
2009-03-03
影响因子：
11.1
作者：
Jakob, B.;Splinter, J.;Taucher-Schoiz, G.
通讯作者：
Taucher-Schoiz, G.
影响因子：
5.3
作者：
CREMER, C;CREMER, T;NAKANISHI, K
通讯作者：
NAKANISHI, K