Division of Protein and Nucleic Acid Chemistry, Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
Division of Protein and Nucleic Acid Chemistry, Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
Mol Cell. 2018 Jun 21;70(6):1067-1080.e12. doi: 10.1016/j.molcel.2018.04.022. Epub 2018 Jun 6.
The replisome must overcome DNA damage to ensure complete chromosome replication. Here, we describe the earliest events in this process by reconstituting collisions between a eukaryotic replisome, assembled with purified proteins, and DNA damage. Lagging-strand lesions are bypassed without delay, leaving daughter-strand gaps roughly the size of an Okazaki fragment. In contrast, leading-strand polymerase stalling significantly impacts replication fork progression. We reveal that the core replisome itself can bypass leading-strand damage by re-priming synthesis beyond it. Surprisingly, this restart activity is rare, mainly due to inefficient leading-strand re-priming, rather than single-stranded DNA exposure or primer extension. We find several unanticipated mechanistic distinctions between leading- and lagging-strand priming that we propose control the replisome's initial response to DNA damage. Notably, leading-strand restart was specifically stimulated by RPA depletion, which can occur under conditions of replication stress. Our results have implications for pathway choice at stalled forks and priming at DNA replication origins.
复制体必须克服 DNA 损伤以确保染色体的完全复制。在这里，我们通过重建用纯化蛋白组装的真核复制体与 DNA 损伤之间的碰撞，描述了这个过程中的最早事件。滞后链的损伤毫不延迟地被绕过，留下大约一个冈崎片段大小的子链缺口。相比之下，领头链聚合酶的停滞严重影响了复制叉的前进。我们揭示了核心复制体本身可以通过在其之外重新启动合成来绕过领头链损伤。令人惊讶的是，这种重新启动的活性很少见，主要是由于领头链重新启动效率低下，而不是单链 DNA 暴露或引物延伸。我们发现了在领头链和滞后链引发之间存在一些出乎意料的机制差异，我们认为这些差异控制着复制体对 DNA 损伤的初始反应。值得注意的是，RPA 耗竭特异性地刺激了领头链的重新启动，而 RPA 耗竭可能发生在复制应激条件下。我们的结果对停滞叉的途径选择和 DNA 复制起点的引发具有重要意义。
