Relationship between DNA double strand break repair and DNA-PK activity

研究了热辐射敏化的机理。双链DNA依赖蛋白激酶（DNA- pk）由DNA- pkcs（催化亚基）和Ku70/80异源二聚体（DNA双链断裂端结合亚基）组成。当DNA双链断裂时，Ku70/80异源二聚体结合到DNA双链断裂端，然后结合DNA- pkcs。我们分析了杂交细胞中DNA-PK活性的热敏性，以及用scid细胞（DNA-PKcs缺陷）提取物恢复DNA-PK活性的可能性。细胞在44℃水浴中热处理。将scid细胞和6 -3细胞的细胞提取物加入热处理后的杂交细胞提取物中，检测DNA-PK活性。当杂交细胞在44℃下加热15 min时，DNA-PK活性降低到检测不到的水平。添加scid细胞提取物后，降低的DNA-PK活性呈浓度依赖性恢复。结果表明，Ku70/80能恢复热失活的DNA-PK，而单独Ku70不能。DNA双链断裂的修复在杂交细胞中分为快速修复和慢速修复两个阶段。而在scid细胞中，DNAdsb是通过单相（缓慢修复）修复的。热处理使快速修复失活。考虑到DNA-PK活性对热敏感，这些结果表明DNA-PK活性维持了快速修复。从这些结果来看，热致敏是DNA-PK热失活的结果。

The mechanism of thermoradiosensitization was studied.Double stranded DNA dependent protein kinase (DNA-PK) consist of DNA-PKcs (catalytic subunit) and Ku70/80 heterodimer (DNA double strand break end binding subunit). When DNA double strand break was occurred, Ku70/80 heterodimer bind to DNA double strand break end and then bind DNA-PKcs. This active form of DNA-PK phosphorylate a variety of proteins including p53, XRCC4 proteins, etc.We analyzed the heat sensitivity of DNA-PK activity in hybrid cells and the possible restoration of this activity with extracts from scid cells (defective in DNA-PKcs). Heat treatment of cells was performed in a water bath at 44℃. The cell extract from scid cells or sxi-3 cells was added to heat-treated hybrid cell extracts, and the DNA-PK activity was assayed. When hybrid cells were heated at 44℃ for 15 min, DNA-PK activity was reduced to undetectable levels. The decreased DNA-PK activity could be restored in concentration-dependent manner with the addition of scid cell extract. This result indicates that Ku70/80, but not Ku70 alone, could restore heat-inactivated DNA-PK.DNA double strand breaks was repaired by two phase, namely fast repair and slow repair in hybrid cells. But in scid cells, DNAdsb was repaired by the single phase (slow repair). The fast repair was inactivated by heat treatment. Considering that DNA-PK activity was heat sensitive, these results indicate that the fast repair was maintained by DNA-PK activity.From these results, thermoradiosensitization was the result of heat inactivation of DNA-PK.