Mårtensson Susanne, Nygren Jonas, Osheroff Neil, Hammarsten Ola
Clinical Chemistry, Gothenburg University, Sahlgrenska University Hospital, 41345 Gothenburg, Sweden.
Radiat Res. 2003 Sep;160(3):291-301. doi: 10.1667/0033-7587(2003)160[0291:aotdpk]2.0.co;2.
The DNA-dependent protein kinase (DNA-PK) is a DNA-end activated protein kinase that is required for efficient repair of DNA double-strand breaks (DSBs) and for normal resistance to ionizing radiation. DNA-PK is composed of a DNA-binding subunit, Ku, and a catalytic subunit, DNA-PKcs (PRKDC). We have previously shown that PRKDC is activated when the enzyme interacts with the terminal nucleotides of a DSB. These nucleotides are often damaged when DSBs are introduced by anticancer agents and could therefore prevent recognition by DNA-PK. To determine whether DNA-PK could recognize DNA strand breaks generated by agents used in the treatment of cancer, we damaged plasmid DNA with anticancer drugs and ionizing radiation. The DNA breaks were tested for the ability to activate purified DNA-PK. The data indicate that DSBs produced by bleomycin, calicheamicin and two types of ionizing radiation ((137)Cs gamma rays and N(7+) ions: high and low linear energy transfer, respectively) activate DNA-PK to levels matching the kinase activation obtained with simple restriction endonuclease-induced DSBs. In contrast, the protein-linked DSBs produced by etoposide and topoisomerase II failed to bind and activate DNA-PK. Our findings indicate that DNA-PK recognizes DSBs regardless of chemical complexity but cannot recognize the protein-linked DSBs produced by etoposide and topoisomerase II.
DNA依赖性蛋白激酶（DNA-PK）是一种DNA末端激活的蛋白激酶，对于DNA双链断裂（DSB）的有效修复以及对电离辐射的正常抗性是必需的。DNA-PK由一个DNA结合亚基Ku和一个催化亚基DNA-PKcs（PRKDC）组成。我们之前已经表明，当该酶与DSB的末端核苷酸相互作用时，PRKDC会被激活。当抗癌药物引入DSB时，这些核苷酸常常会受损，因此可能会阻止DNA-PK的识别。为了确定DNA-PK是否能够识别癌症治疗中使用的药物所产生的DNA链断裂，我们用抗癌药物和电离辐射损伤了质粒DNA。测试了DNA断裂激活纯化的DNA-PK的能力。数据表明，博来霉素、加利车霉素以及两种类型的电离辐射（（137）Csγ射线和N（7+）离子：分别为高和低传能线密度）产生的DSB将DNA-PK激活到与简单限制性内切酶诱导的DSB所获得的激酶激活水平相匹配的程度。相比之下，依托泊苷和拓扑异构酶II产生的蛋白质连接的DSB未能结合并激活DNA-PK。我们的研究结果表明，DNA-PK能够识别DSB，而不论其化学复杂性如何，但不能识别依托泊苷和拓扑异构酶II产生的蛋白质连接的DSB。
