Spatial and temporal cellular responses to single-strand breaks in human cells
Spatial and temporal cellular responses to single-strand breaks in human cells
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DOI：
10.1128/mcb.23.11.3974-3981.2003
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发表时间：
2003-06-01
影响因子：
5.3
通讯作者：
Yasui, R
中科院分区：
文献类型：
--
作者：
Okano, S;Lan, L;Yasui, R
DNA单链断裂（single-strand breaks，SSB）是由活性氧和DNA代谢过程中产生的最常见的DNA损伤之一，但由于缺乏在细胞中单独产生SSB的实验方法，对细胞对SSB的反应的分析仍然困难。通过使用表达外源UV损伤核酸内切酶（UVDE）的人类细胞，并通过膜过滤器中的微孔用UV照射细胞，我们通过UVDE对UV诱导的DNA损伤的直接作用在细胞核的限制区域中产生SSB。通过使用抗体和荧光显微镜表征对SSB的细胞反应。在UV照射后，聚（ADP-核糖）的合成立即发生在照射区域。同时，但依赖于聚（ADP-核糖基）化，XRCC 1从整个细胞核，包括核仁，易位到SSB。XRCC 1蛋白的BRCT 1结构域对于其依赖于聚（ADP-核糖）的募集至SSB是必不可少的。增殖细胞核抗原和染色质组装因子1的p150亚基也以耐洗涤剂的形式积累在SSB，通过抑制聚（ADP-核糖）的合成，其显著减少。我们的研究结果表明，聚（ADP-核糖基）的重要性，在连续的细胞反应SSB。
DNA single-strand breaks (SSB) are one of the most frequent DNA lesions produced by reactive oxygen species and during DNA metabolism, but the analysis of cellular responses to SSB remains difficult due to the lack of an experimental method to produce SSB alone in cells. By using human cells expressing a foreign UV damage endonuclease (UVDE) and irradiating the cells with UV through tiny pores in membrane filters, we created SSB in restricted areas in the nucleus by the immediate action of UVDE on UV-induced DNA lesions. Cellular responses to the SSB were characterized by using antibodies and fluorescence microscopy. Upon UV irradiation, poly(ADP-ribose) synthesis occurred immediately in the irradiated area. Simultaneously, but dependent on poly(ADP-ribosyl)ation, XRCC1 was translocated from throughout the nucleus, including nucleoli, to the SSB. The BRCT1 domain of XRCC1 protein was indispensable for its poly(ADP-ribose)-dependent recruitment to the SSB. Proliferating cell nuclear antigen and the p150 subunit of chromatin assembly factor 1 also accumulated at the SSB in a detergent-resistant form, which was significantly reduced by inhibition of poly(ADP-ribose) synthesis. Our results show the importance of poly(ADP-ribosyl)ation in sequential cellular responses to SSB.