Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint
Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint
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DOI：
10.1101/gad.1301205
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发表时间：
2005-05-01
影响因子：
10.5
通讯作者：
Cimprich, KA
中科院分区：
文献类型：
--
作者：
Byun, TS;Pacek, M;Cimprich, KA
atr依赖的DNA损伤反应途径可以对多种病变以及DNA复制抑制剂作出反应。利用非洲爪蟾卵提取系统，我们发现紫外线照射和顺铂诱导的病变导致MCM解旋酶和DNA聚合酶活性的功能解偶联，这一事件先前在阿飞霉素中被证实。用MCM7或Cdc45（一种推测的解旋酶辅助因子）抑制剂抑制延伸过程中的解偶联，会导致Chk1磷酸化的消除，这表明解偶联是激活检查点所必需的。然而，解偶联并不足以激活检查点，还需要Pol α合成DNA。最后，使用不同大小的质粒，我们证明了在停滞的复制叉上产生的所有未缠绕的DNA都可以促进Chk1磷酸化水平，这表明解耦放大了每个复制叉上的检查点信号。综上所述，这些观察结果表明MCM解旋酶和DNA聚合酶活性的功能解偶联发生在对多种形式的DNA损伤的响应中，并且存在DNA损伤后检查点激活信号产生的一般机制。
The ATR-dependent DNA damage response pathway can respond to a diverse group of lesions as well as inhibitors of DNA replication. Using the Xenopus egg extract system, we show that lesions induced by UV irradiation and cis-platinum cause the functional uncoupling of MCM helicase and DNA polymerase activities, an event previously shown for aphidicolin. Inhibition of uncoupling during elongation with inhibitors of MCM7 or Cdc45, a putative helicase cofactor, results in abrogation of Chk1 phosphorylation, indicating that uncoupling is necessary for activation of the checkpoint. However, uncoupling is not sufficient for checkpoint activation, and DNA synthesis by Pol alpha is also required. Finally, using plasmids of varying size, we demonstrate that all of the unwound DNA generated at a stalled replication fork can contribute to the level of Chk1 phosphorylation, suggesting that uncoupling amplifies checkpoint signaling at each individual replication fork. Taken together, these observations indicate that functional uncoupling of MCM helicase and DNA polymerase activities occurs in response to multiple forms of DNA damage and that there is a general mechanism for generation of the checkpoint-activating signal following DNA damage.