Replication Forks Stalled at Ultraviolet Lesions Are Rescued via RecA and RuvABC Protein-catalyzed Disintegration in Escherichia coli
Replication Forks Stalled at Ultraviolet Lesions Are Rescued via RecA and RuvABC Protein-catalyzed Disintegration in Escherichia coli
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DOI：
10.1074/jbc.m111.322990
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发表时间：
2012-02-24
影响因子：
4.8
通讯作者：
Kuzminov, Andrei
中科院分区：
文献类型：
--
作者：
Khan, Sharik R.;Kuzminov, Andrei
紫外线（UV）照射在亚致死剂量下不会诱导染色体断裂，但紫外线照射会导致遗传不稳定和癌症，这表明染色体是断裂的。在这里，我们表明，紫外线照射诱导亚致死剂量的片段化，但断裂的染色体修复或降解的RecBCD;因此，观察完整的片段化，RecBCD酶需要被灭活。使用定量脉冲场凝胶电泳和敏感的DNA合成测量，我们研究了紫外线辐射诱导的染色体片段化的recBC突变体的机制，比较现有的五种模型的DNA损伤诱导的片段化。我们发现，碎片取决于活性DNA合成之前，但不是之后，紫外线照射。在低紫外线照射剂量下，片段化不需要切除修复或子链缺口修复。片段化完全依赖于RecA催化的同源链交换和RuvABC催化的Holliday连接解析。因此，当复制叉在UV损伤处停滞并退化时，染色体片段化，产生霍利迪连接。值得注意的是，细胞专门利用叉断裂来挽救停滞的复制并避免致命性。
Ultraviolet (UV) irradiation is not known to induce chromosomal fragmentation in sublethal doses, and yet UV irradiation causes genetic instability and cancer, suggesting that chromosomes are fragmented. Here we show that UV irradiation induces fragmentation in sublethal doses, but the broken chromosomes are repaired or degraded by RecBCD; therefore, to observe full fragmentation, RecBCD enzyme needs to be inactivated. Using quantitative pulsed field gel electrophoresis and sensitive DNA synthesis measurements, we investigated the mechanisms of UV radiation-induced chromosomal fragmentation in recBC mutants, comparing five existing models of DNA damage-induced fragmentation. We found that fragmentation depends on active DNA synthesis before, but not after, UV irradiation. At low UV irradiation doses, fragmentation does not need excision repair or daughter strand gap repair. Fragmentation absolutely depends on both RecA-catalyzed homologous strand exchange and RuvABC-catalyzed Holliday junction resolution. Thus, chromosomes fragment when replication forks stall at UV lesions and regress, generating Holliday junctions. Remarkably, cells specifically utilize fork breakage to rescue stalled replication and avoid lethality.