Gadi Sampath Amitash, Hendriks Ivo Alexander, Nielsen Christian Friberg, Popova Petya, Hickson Ian D, Nielsen Michael Lund, Toledo Luis
Center for Chromosome Stability, Institute for Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
Proteomics Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
Mol Cell Proteomics. 2025 Mar;24(3):100915. doi: 10.1016/j.mcpro.2025.100915. Epub 2025 Jan 27.
Accurate genome duplication requires a tightly regulated DNA replication program that relies on the fine regulation of origin firing. While the molecular steps involved in origin firing have been determined predominantly in budding yeast, the complexity of this process in human cells has yet to be fully elucidated. Here, we describe a straightforward proteomics approach to systematically analyze protein recruitment to the chromatin during induced origin firing in human cells. Using a specific inhibitor against CHK1 kinase, we induced a synchronized wave of dormant origin firing (DOF) and assessed the S phase chromatin proteome at different time points. We provide time-resolved loading dynamics of 3269 proteins, including the core replication machinery and origin firing factors. This dataset accurately represents known temporal dynamics of proteins on the chromatin during the activation of replication forks and the subsequent DNA damage due to the hyperactivation of excessive replication forks. Finally, we used our dataset to identify the condensin II subunit NCAPH2 as a novel factor required for efficient origin firing and replication. Overall, we provide a comprehensive resource to interrogate the protein recruitment dynamics of replication origin firing events in human cells.
精确的基因组复制需要一个严格调控的DNA复制程序，该程序依赖于对起始点激活的精细调控。虽然起始点激活所涉及的分子步骤主要是在芽殖酵母中确定的，但这一过程在人类细胞中的复杂性尚未完全阐明。在这里，我们描述了一种直接的蛋白质组学方法，用于系统地分析人类细胞中诱导起始点激活过程中蛋白质向染色质的募集。使用针对CHK1激酶的特异性抑制剂，我们诱导了一波同步的休眠起始点激活（DOF），并在不同时间点评估了S期染色质蛋白质组。我们提供了3269种蛋白质的时间分辨加载动力学，包括核心复制机制和起始点激活因子。该数据集准确地反映了复制叉激活过程中以及由于过多复制叉过度激活导致后续DNA损伤时，蛋白质在染色质上已知的时间动态。最后，我们利用我们的数据集鉴定出凝聚素II亚基NCAPH2是高效起始点激活和复制所需的一个新因子。总体而言，我们提供了一个全面的资源，用于探究人类细胞中复制起始点激活事件的蛋白质募集动力学。
