DNA-PK-independent rejoining of DNA double-strand breaks in human cell extracts in vitro
DNA-PK-independent rejoining of DNA double-strand breaks in human cell extracts in vitro
复制标题
DOI：
10.1080/095530099140825
复制
发表时间：
1999-01-01
影响因子：
2.6
通讯作者：
Iliakis, G
中科院分区：
文献类型：
--
作者：
Cheong, N;Perrault, AR;Iliakis, G
目的：探讨DNA依赖性蛋白激酶（DNA- pk）在电离辐射诱导的DNA双链断裂（dsb）重新连接中的作用。材料和方法：本研究采用先前描述的体外实验，利用由琼脂糖包埋细胞制备的细胞核或“裸”DNA作为底物，S-HeLa细胞提取物作为酶的来源。在这些试验中，dsb的重新连接完全依赖于细胞提取物，并且在最佳反应条件下进行，其程度与在完整细胞中观察到的相似。结果在质粒为基础的实验中证实了dsb的体外再连接。结果：完全抑制DNA-PK活性的wortmannin浓度对dsb在体内的再连接有显著影响，但对dsb在体外的再连接没有影响。此外，使用硫酸铵沉淀法分离细胞提取物，产生能够支持dsb重新连接的蛋白质部分，尽管它们不含有可检测量的DNA-PKcs或Ku80。缺乏DNA-PK活性的MO59J细胞提取物也能有效地在体外重新连接dsb。最后，在基于质粒的实验中，dsb的重新连接不受wortmannin的影响，并且也可以用MO59J细胞的提取物检测到。结论：这些发现与遗传学研究相反，遗传学研究表明DNA-PK活性是dsb在体内有效重新连接的必要条件。体外和体内结果之间的差异可能不归因于染色质结构，因为wortmannin在使用细胞核作为底物时没有影响。据推测，体内和体外结果之间的差异可以通过假设dsb再连接中有多种途径的运作来解释，其中一些不需要DNA-PK，或者假设DNA-PK在完整细胞中仅具有调节/损伤感知作用，而不直接参与dsb再连接。
Purpose: To investigate the role of DNA-dependent protein kinase (DNA-PK) in the rejoining of ionizing radiation-induced DNA double-strand breaks (dsb).Materials and methods: This study employed previously described in vitro assays that utilize nuclei or 'naked' DNA prepared from agarose-embedded cells as a substrate and S-HeLa cell extracts as a source of enzymes. Rejoining of dsb in these assays is absolutely dependent on cell extract and it proceeds, under optimal reaction conditions, to an extent similar to that observed in intact cells. Results were confirmed in a plasmid-based assay for in vitro rejoining of dsb.Results: It is shown that concentrations of wortmannin completely inhibiting DNA-PK activity profoundly affect the rejoining df dsb in vivo, but have no effect on dsb rejoining in vitro. Furthermore, fractionation of cell extracts using ammonium sulphate precipitation, generates protein fractions that are able to support dsb rejoining, despite the fact that they do not contain detectable amounts of either DNA-PKcs or Ku80. Efficient rejoining of dsb in vitro is also observed with extracts of MO59J cells that lack DNA-PK activity. Finally, rejoining of dsb remains unaffected by wortmannin in a plasmid-based assay, and is also detectable with extracts of MO59J cells.Conclusions: These findings are in contrast with genetic studies demonstrating a requirement for DNA-PK activity for efficient rejoining of dsb in vivo. The difference between in vitro and in vivo results may not be attributed to chromatin structure since wortmannin was without an effect when using nuclei as a substrate. It is speculated that the differences between in vivo and in vitro results can be explained either by assuming the operation of multiple pathways in dsb rejoining, some of which do not require DNA-PK, or by postulating a purely regulatory/damage sensing role for DNA-PK in intact cells but no direct involvement in dsb rejoining.