The Novo Nordisk Center for Protein Research (CPR), Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark; Biotech Research and Innovation Centre (BRIC), Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark.
Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3BF, UK.
Mol Cell. 2021 Mar 4;81(5):1084-1099.e6. doi: 10.1016/j.molcel.2020.12.025. Epub 2021 Jan 14.
Cells have evolved an elaborate DNA repair network to ensure complete and accurate DNA replication. Defects in these repair machineries can fuel genome instability and drive carcinogenesis while creating vulnerabilities that may be exploited in therapy. Here, we use nascent chromatin capture (NCC) proteomics to characterize the repair of replication-associated DNA double-strand breaks (DSBs) triggered by topoisomerase 1 (TOP1) inhibitors. We reveal profound changes in the fork proteome, including the chromatin environment and nuclear membrane interactions, and identify three classes of repair factors according to their enrichment at broken and/or stalled forks. ATM inhibition dramatically rewired the broken fork proteome, revealing that ataxia telangiectasia mutated (ATM) signalling stimulates DNA end resection, recruits PLK1, and concomitantly suppresses the canonical DSB ubiquitination response by preventing accumulation of RNF168 and BRCA1-A. This work and collection of replication fork proteomes provide a new framework to understand how cells orchestrate homologous recombination repair of replication-associated DSBs.
细胞已经进化出了一个复杂的 DNA 修复网络，以确保 DNA 复制的完整和准确。这些修复机制的缺陷会导致基因组不稳定并促进癌变，同时产生可能在治疗中被利用的弱点。在这里，我们使用新生染色质捕获（NCC）蛋白质组学来描述拓扑异构酶 1（TOP1）抑制剂引发的复制相关 DNA 双链断裂（DSB）的修复。我们揭示了叉状结构蛋白质组的深刻变化，包括染色质环境和核膜相互作用，并根据它们在断裂和/或停滞叉上的富集程度，将修复因子分为三类。ATM 抑制作用显著改变了断裂叉状结构蛋白质组，表明共济失调毛细血管扩张突变（ATM）信号刺激 DNA 末端切除，募集 PLK1，并通过防止 RNF168 和 BRCA1-A 的积累来同时抑制经典的 DSB 泛素化反应。这项工作和复制叉状结构蛋白质组的收集为理解细胞如何协调复制相关 DSB 的同源重组修复提供了一个新的框架。
