Checkpoint signaling from a single DNA interstrand crosslink.
Checkpoint signaling from a single DNA interstrand crosslink.
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DOI：
10.1016/j.molcel.2009.08.014
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发表时间：
2009-09-11
期刊：
影响因子：
16
通讯作者：
Gautier J
中科院分区：
文献类型：
--
作者：
Ben-Yehoyada M;Wang LC;Kozekov ID;Rizzo CJ;Gottesman ME;Gautier J
DNA链间交联(ICL)是丝裂霉素C和顺铂等化疗药物引起的毒性最大的损伤。通过共价连接两条DNA链，ICL可以防止DNA熔化、转录和复制。关于ICL信号和修复的研究一直受到限制，因为这些药物会产生额外的DNA损伤，从而触发检查点信号。在这里，我们监测单个位点特异性ICL的感知、信号传递和修复，这些ICL来自非洲爪哇卵子和哺乳动物细胞的无细胞提取物。值得注意的是，我们证明ICL触发的检查点反应独立于起源启动的DNA复制和DNA聚合酶和DNA解旋酶的解偶联。Fanconi贫血途径作用于RPA-ATR-Chk1上游，产生ICL信号。该系统还在涉及广泛、无错误的DNA合成的反应中修复ICL。修复通过起源依赖和起源非依赖两种机制发生。我们的数据表明，细胞对交联剂的敏感性是由检查点和DNA修复缺陷造成的。
DNA interstrand crosslinks (ICLs) are the most toxic lesions induced by chemotherapeutic agents such as Mitomycin C and Cisplatin. By covalently linking both DNA strands, ICLs prevent DNA melting, transcription, and replication. Studies on ICL signaling and repair have been limited because these drugs generate additional DNA lesions that trigger checkpoint signaling. Here, we monitor sensing, signaling from and repairing of a single, site-specific ICL in cell-free extract derived from Xenopus eggs and in mammalian cells. Notably, we demonstrate that ICLs trigger a checkpoint response independently of origin-initiated DNA replication and uncoupling of DNA polymerase and DNA helicase. The Fanconi anemia pathway acts upstream of RPA-ATR-Chk1 to generate the ICL signal. The system also repairs ICLs in a reaction that involves extensive, error-free, DNA synthesis. Repair occurs by both origin-dependent and origin-independent mechanisms. Our data suggest that cell sensitivity to crosslinking agents results from both checkpoint and DNA repair defects.
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