Department of Genetics, Microbiology and Toxicology, Arrhenius Laboratories of Natural Science, Stockholm University, S-106 91 Stockholm, Sweden.
DNA Repair (Amst). 2012 Dec 1;11(12):976-85. doi: 10.1016/j.dnarep.2012.09.010. Epub 2012 Oct 23.
DNA interstrand crosslinks (ICLs) are highly toxic lesions that covalently link both strands of DNA and distort the DNA helix. Crosslinking agents have been shown to stall DNA replication and failure to repair ICL lesions before encountered by replication forks may induce severe DNA damage. Most knowledge of the ICL repair process has been revealed from studies in bacteria and cell extracts. However, for mammalian cells the process of ICL repair is still unclear and conflicting data exist. In this study we have explored the fate of psoralen-induced ICLs during replication, by employing intact mammalian cells and novel techniques. By comparative studies distinguishing between effects by monoadducts versus ICLs, we have been able to link the block of replication to the ICLs induction. We found that the replication fork was equally blocked by ICLs in wild-type cells as in cells deficient in ERCC1/XPF and XRCC3. The formation of ICL induced double strand breaks (DSBs), detected by formation of 53PB1 foci, was equally induced in the three cell lines suggesting that these proteins are involved at a later step of the repair process. Furthermore, we found that forks blocked by ICLs were neither bypassed, restarted nor restored for several hours. We propose that this process is different from that taking place following monoadduct induction by UV-light treatment where replication bypass is taking place as an early step. Altogether our findings suggest that restoration of an ICL blocked replication fork, likely initiated by a DSB occurs relatively rapidly at a stalled fork, is followed by restoration, which seems to be a rather slow process in intact mammalian cells.
DNA 链间交联（ICLs）是一种高度有毒的损伤，它会使 DNA 的两条链发生共价交联，并使 DNA 螺旋扭曲。交联剂已被证明会使 DNA 复制停滞，如果在复制叉遇到之前不能修复 ICL 损伤，可能会引起严重的 DNA 损伤。大多数关于 ICL 修复过程的知识都是通过细菌和细胞提取物的研究揭示的。然而，对于哺乳动物细胞，ICL 修复过程仍不清楚，并且存在相互矛盾的数据。在这项研究中，我们通过使用完整的哺乳动物细胞和新的技术，探索了吖啶酮诱导的 ICL 在复制过程中的命运。通过区分单加合物和 ICL 效应的比较研究，我们能够将复制的阻断与 ICL 的诱导联系起来。我们发现，在野生型细胞中，ICLs 同样会阻断复制叉，而在 ERCC1/XPF 和 XRCC3 缺陷型细胞中也是如此。通过形成 53PB1 焦点来检测 ICL 诱导的双链断裂（DSBs），在这三种细胞系中同样被诱导，这表明这些蛋白参与了修复过程的后期步骤。此外，我们发现 ICL 阻断的叉不能在几个小时内被绕过、重新启动或恢复。我们提出，这个过程与 UV 光处理诱导单加合物后发生的过程不同，在 UV 光处理诱导单加合物后，复制绕过是一个早期步骤。总之，我们的研究结果表明，在停滞的复制叉上，修复 ICL 阻断的复制叉的过程相对较快，随后是修复，在完整的哺乳动物细胞中，修复似乎是一个相当缓慢的过程。
