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首页> 外文期刊>Molecular and Cellular Biology >The Structure-Specific Endonuclease Ercc1-Xpf Is Required To Resolve DNA Interstrand Cross-Link-Induced Double-Strand Breaks
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The Structure-Specific Endonuclease Ercc1-Xpf Is Required To Resolve DNA Interstrand Cross-Link-Induced Double-Strand Breaks

机译：需要特定于结构的核酸内切酶Ercc1-Xpf以解决DNA链间交联引起的双链断裂。

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Interstrand cross-links (ICLs) are an extremely toxic class of DNA damage incurred during normal metabolism or cancer chemotherapy. ICLs covalently tether both strands of duplex DNA, preventing the strand unwinding that is essential for polymerase access. The mechanism of ICL repair in mammalian cells is poorly understood. However, genetic data implicate the Ercc1-Xpf endonuclease and proteins required for homologous recombination-mediated double-strand break (DSB) repair. To examine the role of Ercc1-Xpf in ICL repair, we monitored the phosphorylation of histone variant H2AX (γ-H2AX). The phosphoprotein accumulates at DSBs, forming foci that can be detected by immunostaining. Treatment of wild-type cells with mitomycin C (MMC) induced γ-H2AX foci and increased the amount of DSBs detected by pulsed-field gel electrophoresis. Surprisingly, γ-H2AX foci were also induced in Ercc1?/? cells by MMC treatment. Thus, DSBs occur after cross-link damage via an Ercc1-independent mechanism. Instead, ICL-induced DSB formation required cell cycle progression into S phase, suggesting that DSBs are an intermediate of ICL repair that form during DNA replication. In Ercc1?/? cells, MMC-induced γ-H2AX foci persisted at least 48 h longer than in wild-type cells, demonstrating that Ercc1 is required for the resolution of cross-link-induced DSBs. MMC triggered sister chromatid exchanges in wild-type cells but chromatid fusions in Ercc1?/? and Xpf mutant cells, indicating that in their absence, repair of DSBs is prevented. Collectively, these data support a role for Ercc1-Xpf in processing ICL-induced DSBs so that these cytotoxic intermediates can be repaired by homologous recombination.
机译：链间交联（ICL）是正常代谢或癌症化学疗法中引起的一种极毒性的DNA损伤。 ICL与双链DNA的两条链共价连接，从而防止了聚合酶访问必不可少的链解绕。对ICL修复哺乳动物细胞的机制了解甚少。但是，遗传数据暗示了Ercc1-Xpf核酸内切酶和同源重组介导的双链断裂（DSB）修复所需的蛋白质。为了检查Ercc1-Xpf在ICL修复中的作用，我们监测了组蛋白变体H2AX（γ-H2AX）的磷酸化。磷蛋白在DSB处积聚，形成可以通过免疫染色检测到的病灶。用丝裂霉素C（MMC）处理野生型细胞诱导的γ-H2AX病灶，并增加了通过脉冲场凝胶电泳检测到的DSB的数量。出乎意料的是，通过MMC处理在 Ercc1 ？/？细胞中也诱导了γ-H2AX病灶。因此，DSB在通过Ercc1独立机制发生交联破坏后发生。相反，ICL诱导的DSB形成需要细胞周期进入S期，这表明DSB是DNA复制过程中形成的ICL修复的中间产物。在 Ercc1 ？/？细胞中，MMC诱导的γ-H2AX病灶比野生型细胞持续至少48小时长的时间，这表明需要Ercc1才能解析交联诱导的DSB。 MMC触发了野生型细胞中的姐妹染色单体交换，但 Ercc1 ？/？和Xpf突变细胞中的染色单体融合，表明在它们不存在的情况下，DSB的修复被阻止。总体而言，这些数据支持Ercc1-Xpf在处理ICL诱导的DSB中的作用，以便可以通过同源重组修复这些细胞毒性中间体。

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