Trenz Kristina, Smith Eloise, Smith Sarah, Costanzo Vincenzo
Genome Stability Unit, London Research Institute, Clare Hall Laboratories, South Mimms, London, UK.
EMBO J. 2006 Apr 19;25(8):1764-74. doi: 10.1038/sj.emboj.7601045. Epub 2006 Apr 6.
Ataxia-telangiectasia mutated (ATM), ataxia-telangiectasia Rad3-related (ATR) and the Mre11/Rad50/Nbs1 complex ensure genome stability in response to DNA damage. However, their essential role in DNA metabolism remains unknown. Here we show that ATM and ATR prevent accumulation of DNA double-strand breaks (DSBs) during chromosomal replication. Replicating chromosomes accumulate DSBs in Xenopus laevis egg extracts depleted of ATM and ATR. Addition of ATM and ATR proteins to depleted extracts prevents DSB accumulation by promoting restart of collapsed replication forks that arise during DNA replication. We show that collapsed forks maintain MCM complex but lose Pol epsilon, and that Pol epsilon reloading requires ATM and ATR. Replication fork restart is abolished in Mre11 depleted extracts and is restored by supplementation with recombinant human Mre11/Rad50/Nbs1 complex. Using a novel fluorescence resonance energy transfer-based technique, we demonstrate that ATM and ATR induce Mre11/Rad50/Nbs1 complex redistribution to restarting forks. This study provides direct biochemical evidence that ATM and ATR prevent accumulation of chromosomal abnormalities by promoting Mre11/Rad50/Nbs1 dependent recovery of collapsed replication forks.
共济失调毛细血管扩张症突变基因（ATM）、共济失调毛细血管扩张症Rad3相关基因（ATR）以及Mre11/Rad50/Nbs1复合物可确保细胞基因组在应对DNA损伤时的稳定性。然而，它们在DNA代谢中的重要作用仍不清楚。在此，我们发现ATM和ATR可防止染色体复制过程中DNA双链断裂（DSB）的积累。在缺乏ATM和ATR的非洲爪蟾卵提取物中，正在复制的染色体积累了DSB。向耗尽提取物中添加ATM和ATR蛋白，可通过促进DNA复制过程中出现的崩溃复制叉重新启动来防止DSB积累。我们发现，崩溃的复制叉保留了MCM复合物，但失去了聚合酶ε，并且聚合酶ε重新加载需要ATM和ATR。在缺乏Mre11的提取物中，复制叉重新启动被消除，补充重组人Mre11/Rad50/Nbs1复合物可恢复这一过程。使用一种基于荧光共振能量转移的新技术，我们证明ATM和ATR可诱导Mre11/Rad50/Nbs1复合物重新分布到重新启动的复制叉上。这项研究提供了直接的生化证据，表明ATM和ATR通过促进依赖Mre11/Rad50/Nbs1的崩溃复制叉恢复来防止染色体异常的积累。
