The ATR replication checkpoint ensures that stalled forks remain stable when replisome movement is impeded. Using an improved iPOND protocol combined with SILAC mass spectrometry, we characterized human replisome dynamics in response to fork stalling. Our data provide a quantitative picture of the replisome and replication stress response proteomes in 32 experimental conditions. Importantly, rather than stabilize the replisome, the checkpoint prevents two distinct types of fork collapse. Unsupervised hierarchical clustering of protein abundance on nascent DNA is sufficient to identify protein complexes and place newly identified replisome-associated proteins into functional pathways. As an example, we demonstrate that ZNF644 complexes with the G9a/GLP methyltransferase at replication forks and is needed to prevent replication-associated DNA damage. Our data reveal how the replication checkpoint preserves genome integrity, provide insights into the mechanism of action of ATR inhibitors, and will be a useful resource for replication, DNA repair, and chromatin investigators.
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