DNA-dependent protein kinase stimulates an independently active, nonhomologous, end-joining apparatus.
DNA-dependent protein kinase stimulates an independently active, nonhomologous, end-joining apparatus.
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发表时间：
2000-03
期刊：
影响因子：
11.2
通讯作者：
S. DiBiase;Z. Zeng;Richard Chen;T. Hyslop;W. Curran;G. Iliakis
中科院分区：
文献类型：
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作者：
S. DiBiase;Z. Zeng;Richard Chen;T. Hyslop;W. Curran;G. Iliakis
通过非同源末端连接（NHEJ）可以有效地去除高等真核生物DNA中的双链断裂（DSB）。遗传学研究表明，NHEJ中存在DNA依赖性蛋白激酶（DNA-PK），但这种蛋白复合物在重接反应中的确切功能仍有待阐明。我们比较了人神经胶质瘤细胞系M059-J（缺乏DNA-PK的催化亚基（DNA-PKcs））和它们的同基因但DNA-PK熟练的对应物M059-K中DNA DSB的重新连接。在这两种细胞系中，DNA DSB的重新连接是双相的，其中快速部分和缓慢部分的半衰期分别约为22分钟和12小时。DNA-PK活性的缺乏并没有改变这些重新连接的组件的半衰期，但从17%增加到72%的DNA DSB重新连接的比例与缓慢的动力学。DNA DSB重连接在两种细胞系中几乎完成，并且与M059-K细胞相比，孵育30 h后M059-J中未连接断裂的数量仅略有增加。Wortmannin对杀死M059-K细胞具有放射增敏作用，并强烈抑制DNA DSB再连接。渥曼青霉素不影响杀伤的放射敏感性，并且仅对M059-J细胞中的DNA DSB重新连接产生适度抑制，这表明对于这些终点，DNA-PK是药物的主要靶点。这些观察结果表明，DNA-PK缺陷极大地降低了DNA DSB以快速动力学重新连接的比例，但对剩余未连接的部分影响很小。我们提出，在高等真核生物中，一个进化上保守的，独立活跃的，但固有的缓慢NHEJ途径被DNA-PKcs刺激30倍，以迅速从基因组中去除DNA DSB。预期刺激是局部性质的，并且DNA DSB附近DNA-PKcs的存在决定了重新连接是否遵循快速或缓慢的动力学。DNA-PKcs的结构和调节功能可能介导DNA DSB重新连接的这种令人印象深刻的加速，并且在这个大蛋白的一定范围内的染色质区域可能从这些活动中受益。我们提出术语DNA-PK监测域来描述这些区域。
Double-strand breaks (DSBs) can be efficiently removed from the DNA of higher eukaryotes by nonhomologous end-joining (NHEJ). Genetic studies implicate the DNA-dependent protein kinase (DNA-PK) in NHEJ, but the exact function of this protein complex in the rejoining reaction remains to be elucidated. We compared rejoining of DNA DSBs in a human glioma cell line, M059-J, lacking the catalytic subunit of DNA-PK (DNA-PKcs), and their isogenic but DNA-PK-proficient counterpart, M059-K. In both cell lines, rejoining of DNA DSBs was biphasic, with a fast and a slow component operating with a half-life of approximately 22 min and 12 h, respectively. Deficiency in DNA-PK activity did not alter the half-times of either of these components of rejoining but increased from 17 to 72% the proportion of DNA DSB rejoining with slow kinetics. DNA DSB rejoining was nearly complete in both cell lines, and there was only a small increase in the number of unrejoined breaks in M059-J as compared with M059-K cells after 30 h of incubation. Wortmannin radiosensitized to killing M059-K cells and strongly inhibited DNA DSB rejoining. Wortmannin did not affect the radiosensitivity to killing and produced only a modest inhibition in DNA DSB rejoining in M059-J cells, suggesting that, for these end points, DNA-PK is the principal target of the drug. These observations demonstrate that DNA-PK deficiency profoundly decreases the proportion of DNA DSB rejoining with fast kinetics but has only a small effect on the fraction remaining unrejoined. We propose that in higher eukaryotes, an evolutionarily conserved, independently active, but inherently slow NHEJ pathway is stimulated 30-fold by DNA-PKcs to rapidly remove DNA DSBs from the genome. The stimulation is expected to be of local nature and the presence of DNA-PKcs in the vicinity of the DNA DSB determines whether rejoining will follow fast or slow kinetics. Structural and regulatory functions of DNA-PKcs may mediate this impressive acceleration of DNA DSB rejoining, and regions of chromatin within a certain range from this large protein may benefit from these activities. We propose the term DNA-PK surveillance domains to describe these regions.