In situ analysis of repair processes for oxidative DNA damage in mammalian cells
In situ analysis of repair processes for oxidative DNA damage in mammalian cells
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DOI：
10.1073/pnas.0406048101
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发表时间：
2004-09-21
影响因子：
11.1
通讯作者：
Yasui, A
中科院分区：
文献类型：
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作者：
Lan, L;Nakajima, S;Yasui, A
氧化性DNA损伤导致转录和复制的阻滞和错误，导致细胞死亡和基因组不稳定。虽然损伤的修复机制已经在体外进行了广泛的分析，但实际的体内修复过程在很大程度上仍然未知。在这里，通过显微镜透镜用UVA激光照射，我们有条件地在哺乳动物细胞的限制性核区域产生单链断裂和氧化性碱基损伤。我们通过使用抗体和GFP标记的蛋白质在照射后的真实的时间内显示了人类细胞中氧化DNA损伤的快速有序的DNA修复过程。此外，我们通过使用修复缺陷的哺乳动物细胞表征了修复途径，并发现DNA聚合酶13分别通过其31和8 kDa结构域在单链断裂和氧化碱基损伤处积累，并且XRCC1对于聚合酶β依赖性和增殖细胞核抗原依赖性的单链断裂修复途径都是必需的。因此，氧化性DNA损伤的修复是基于在活细胞中DNA损伤位点处操作的各种蛋白质之间的时间和功能相互作用。
Oxidative DNA damage causes blocks and errors in transcription and replication, leading to cell death and genomic instability. Although repair mechanisms of the damage have been extensively analyzed in vitro, the actual in vivo repair processes remain largely unknown. Here, by irradiation with an UVA laser through a microscope lens, we have conditionally produced single-strand breaks and oxidative base damage at restricted nuclear regions of mammalian cells. We showed, in real time after irradiation by using antibodies and GFP-tagged proteins, rapid and ordered DNA repair processes of oxidative DNA damage in human cells. Furthermore, we characterized repair pathways by using repair-defective mammalian cells and found that DNA polymerase 13 accumulated at single-strand breaks and oxidative base damage by means of its 31- and 8-kDa domains, respectively, and that XRCC1 is essential for both polymerarse beta-dependent and proliferating cell nuclear antigen-dependent repair pathways of single-strand breaks. Thus, the repair of oxidative DNA damage is based on temporal and functional interactions among various proteins operating at the site of DNA damage in living cells.