Proteome dynamics at stalled replication forks reveal a coordinated recruitment of repair factors

The faithful replication of DNA is a fundamental cellular process that ensures the transfer of genetic information during cell division. Genome duplication takes place during S phase and requires the dynamic and coordinated recruitment of multiple proteins at the replication forks. The instability and collapse of the replication fork can occur when cells are exposed to different genotoxic stresses, which can be overcome by the action of DNA repair pathways. Studying the proteomic interactions implicated in those events is thus a crucial step in understanding the complex biological mechanisms involved in DNA replication and repair. Proximity-dependent biotinylation coupled with mass spectrometry identification of proteins (BioID2) was used on 17 proteins from four different complexes known to play a role during DNA replication and repair to study the assembly and recruitment of proteins at stalled replication forks caused by treatment with hydroxyurea. This analysis revealed a vast interaction network of 108 proteins modulated in the presence of hydroxyurea; 45 being enriched and 63 depleted. Interestingly, 11 of them were found to be both enriched and depleted depending on the complex considered, suggesting a dynamic reorganization of the players and their interactions (?). Furthermore, the analysis identified several poorly characterized proteins, thus uncovering new players in the cellular response to DNA replication arrest. Overall, this collection of replication fork proteomes enables large-scale identification of the mechanisms of assembly and disassembly of protein complexes at the sites of stalled replication forks and provides a new framework to understand how cells respond to obstacles on DNA template, causing fork stalling or collapse.

DNA保真性复制是一项基础细胞过程，可保障细胞分裂时期遗传信息的稳定传递。基因组复制发生于细胞周期S期，需要多种蛋白质在复制叉处被动态且协同地招募。当细胞暴露于多种基因毒性应激条件下时，复制叉可发生不稳定甚至崩解，而DNA修复通路可介导此类损伤的修复。因此，研究与上述事件相关的蛋白质组相互作用，是解析DNA复制与修复所涉及的复杂生物学机制的关键步骤。本研究采用邻近依赖型生物素标记联合蛋白质质谱鉴定技术（Proximity-dependent biotinylation coupled with mass spectrometry identification of proteins, BioID2），对已知参与DNA复制与修复过程的4种复合物中的17种蛋白质进行标记检测，以探究羟基脲处理诱导停滞的复制叉处的蛋白质组装与招募机制。本次分析共鉴定得到108种受羟基脲调控的蛋白质相互作用网络，其中45种蛋白质表达富集，63种蛋白质表达耗竭。有趣的是，其中11种蛋白质的表达丰度会因所分析的复合物不同而同时呈现富集与耗竭两种状态，这表明蛋白质组分及其相互作用存在动态重排（？）。此外，本次分析还鉴定出多种功能尚未被充分阐明的蛋白质，从而揭示了参与DNA复制停滞细胞应答过程的全新组分。总体而言，本数据集收录的复制叉蛋白质组数据，可实现对停滞复制叉处蛋白质复合物组装与解离机制的大规模解析，并为理解细胞如何应对DNA模板上的障碍（进而引发复制叉停滞或崩解）提供了全新的研究框架。