PDF(Pubmed)
Abstract：
DNA replication is a fundamental cellular process that ensures the transfer of genetic information during cell division. Genome duplication takes place in S phase and requires a dynamic and highly coordinated recruitment of multiple proteins at replication forks. Various genotoxic stressors lead to fork instability and collapse, hence the need for DNA repair pathways. By identifying the multitude of protein interactions implicated in those events, we can better grasp the complex and dynamic molecular mechanisms that facilitate DNA replication and repair. Proximity-dependent biotin identification was used to identify associations with 17 proteins within four core replication components, namely the CDC45/MCM2-7/GINS helicase that unwinds DNA, the DNA polymerases, replication protein A subunits, and histone chaperones needed to disassemble and reassemble chromatin. We further investigated the impact of genotoxic stress on these interactions. This analysis revealed a vast proximity association network with 108 nuclear proteins further modulated in the presence of hydroxyurea; 45 being enriched and 63 depleted. Interestingly, hydroxyurea treatment also caused a redistribution of associations with 11 interactors, meaning that the replisome is dynamically reorganized when stressed. The analysis identified several poorly characterized proteins, thereby uncovering new putative players in the cellular response to DNA replication arrest. It also provides a new comprehensive proteomic framework to understand how cells respond to obstacles during DNA replication.
摘要：
DNA复制是确保细胞分裂过程中遗传信息转移的基本细胞过程。基因组复制发生在S期，需要在复制叉处动态且高度协调地募集多种蛋白质。各种基因毒性应激导致叉子不稳定和崩溃，因此需要DNA修复途径。通过鉴定与这些事件有关的多种蛋白质相互作用，我们可以更好地掌握促进DNA复制和修复的复杂和动态分子机制。邻近依赖性生物素鉴定（BioID）用于鉴定与四种核心复制成分中的17种蛋白质的关联。即解绕DNA的CDC45/MCM2-7/GINS（CMG）解旋酶，DNA聚合酶，复制蛋白A亚基，和组蛋白伴侣需要拆卸和重新组装染色质。我们进一步研究了基因毒性应激对这些相互作用的影响。该分析揭示了广泛的邻近关联网络，在羟基脲存在下进一步调节了108个核蛋白；富集45个，耗尽63个。有趣的是,羟基脲处理还导致了与11个相互作用者的关联的重新分配，这意味着当压力时，复制体会动态重组。分析确定了几种特征不佳的蛋白质，从而在细胞对DNA复制停滞的反应中发现新的推定参与者。它还提供了一个新的全面的蛋白质组学框架，以了解细胞在DNA复制过程中如何应对障碍。
