As its name implies, the DNA dependent protein kinase (DNA-PK) requires DNA double-stranded ends for enzymatic activation. Here, I demonstrate that hairpinned DNA ends are ineffective for activating the kinase toward many of its well-studied substrates (p53, XRCC4, XLF, HSP90). However, hairpinned DNA ends robustly stimulate certain DNA-PK autophosphorylations. Specifically, autophosphorylation sites within the ABCDE cluster are robustly phosphorylated when DNA-PK is activated by hairpinned DNA ends. Of note, phosphorylation of the ABCDE sites is requisite for activation of the Artemis nuclease that associates with DNA-PK to mediate hairpin opening. This finding suggests a multi-step mechanism of kinase activation. Finally, I find that all non-homologous end joining (NHEJ) defective cells (whether deficient in components of the DNA-PK complex or components of the ligase complex) are similarly deficient in joining DNA double-stranded breaks (DSBs) with hairpinned termini.

中文翻译：

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发夹 DNA 末端对 DNA-PK 的激活揭示了激酶激活的逐步机制。

顾名思义，DNA 依赖性蛋白激酶 (DNA-PK) 需要 DNA 双链末端来进行酶促活化。在这里，我证明发夹 DNA 末端对于激活激酶对许多经过充分研究的底物（p53、XRCC4、XLF、HSP90）无效。然而，发夹 DNA 末端强烈刺激某些 DNA-PK 自磷酸化。具体来说，当 DNA-PK 被发夹的 DNA 末端激活时，ABCDE 簇内的自磷酸化位点被强烈磷酸化。值得注意的是，ABCDE 位点的磷酸化是激活与 DNA-PK 相关以介导发夹开放的 Artemis 核酸酶所必需的。这一发现表明激酶激活的多步骤机制。最后，