Suppression of the poly(ADP-ribose) polymerase activity by DNA-dependent protein kinase in vitro
Suppression of the poly(ADP-ribose) polymerase activity by DNA-dependent protein kinase in vitro
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DOI：
10.1038/sj.onc.1202823
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发表时间：
1999-08-12
期刊：
影响因子：
8
通讯作者：
Noda, M
中科院分区：
文献类型：
--
作者：
Ariumi, Y;Masutani, M;Noda, M
DNA依赖性蛋白激酶（DNA-PK）是DNA双链断裂修复的重要组成部分。然而，DNA-PK作用的机制尚未完全理解。聚ADP-核糖聚合酶（Poly（ADP-ribose）polymerase，PARP）是另一种对DNA末端具有高亲和力的核酶。在这项研究中，我们分析了这些tao酶之间的相互作用。首先，发现DNA-PK抑制PARP活性并改变聚（ADP-核糖基）化的模式。尽管DNA-PK以DNA依赖性方式磷酸化PARP，但这种修饰不太可能导致PARP活性的抑制，因为这种抑制即使在不存在ATP的情况下也会发生。相反，PARP被发现在体外ADP-核糖基化DNA-PK。然而，DNA-PK的自磷酸化活性不受此修饰的影响。在竞争性电泳迁移率变动分析中，发现DNA-PK的DNA结合组分Ku 70/80复合物对DNA短片段的亲和力高于PARP。此外，免疫共沉淀分析表明Ku和PARP之间存在直接或密切的关联。因此，DNA-PK抑制PARP活性，可能是通过直接结合和/或螯合DNA末端，DNA末端作为两种酶的重要刺激物。
It has been suggested that DNA-dependent protein kinase (DNA-PK) is a central component of DNA double-strand-break repair. The mechanism of DNA-PK action, however, has not been fully understood. Poly(ADP-ribose) polymerase (PARP) is another nuclear enzyme which has high affinity to DNA ends. In this study, we analysed the interaction between these tao enzymes. First, DNA-PK was found to suppress the PARP activity and alters the pattern of poly(ADP-ribosyl)ation. Although DNA-PK phosphorylates PARP in a DNA-dependent manner, this modification is unlikely to be responsible for the suppression of PARP activity, since this suppression occurs even in the absence of ATP. Conversely, PARP was found to ADP-ribosylate DNA-PK in vitro. However, the autophosphorylation activity of DNA-PK was not influenced by this modification. In a competitive electrophoretic mobility shift assay, Ku 70/80 complex, the DNA binding component of DNA-PK, was found to have higher affinity to a short fragment of DNA than does PARP. Furthermore, co-immunoprecipitation analysis suggested direct or close association between Ku and PARP. Thus, DNA-PK suppresses PARP activity, probably through direct binding and/or sequestration of DNA-ends which serve as an important stimulator for both enzymes.