Summary 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.

Background: DNA replication and the replication stress response require the coordinated actions of many proteins. Results: iPOND coupled with mass spectrometry identified 290 proteins associated with active, stalled, or collapsed replication forks. Conclusion: iPOND-MS is a useful discovery tool. Significance: The data increase our understanding of the network of proteins involved in DNA replication and the replication stress response.

The ATR checkpoint kinase coordinates cellular responses to DNA replication stress. Budding yeast contain three activators of Mec1 (the ATR orthologue); however, only TOPBP1 is known to activate ATR in vertebrates. We identified ETAA1 as a replication stress response protein in two proteomic screens. ETAA1-deficient cells accumulate double-strand breaks, sister chromatid exchanges, and other hallmarks of genome instability. They are also hyper-sensitive to replication stress and have increased frequencies of replication fork collapse. ETAA1 contains two RPA-interaction motifs that localize ETAA1 to stalled replication forks. It also interacts with several DNA damage response proteins including the BLM/TOP3α/RMI1/RMI2 and ATR/ATRIP complexes. It binds ATR/ATRIP directly using a motif with sequence similarity to the TOPBP1-ATR activation domain; and like TOPBP1, ETAA1 acts as a direct ATR activator. ETAA1 functions in parallel to the TOPBP1/RAD9/HUS1/RAD1 pathway to regulate ATR and maintain genome stability. Thus, vertebrate cells contain at least two ATR activating proteins.

SUMMARY RAD51 promotes homology-directed repair (HDR), replication fork reversal, and stalled fork protection. Defects in these functions cause genomic instability and tumorigenesis, but also generate hypersensitivity to cancer therapeutics. Here we describe the identification of RADX as an RPA-like, single-strand DNA binding protein. RADX is recruited to replication forks where it prevents fork collapse by regulating RAD51. When RADX is inactivated, excessive RAD51 activity slows replication elongation and causes double-strand breaks. In cancer cells lacking BRCA2, RADX deletion restores fork protection without restoring HDR. Furthermore, RADX inactivation confers chemotherapy and PARP inhibitor resistance to cancer cells with reduced BRCA2/RAD51 pathway function. By antagonizing RAD51 at forks, RADX allows cells to maintain a high capacity for HDR while ensuring that replication functions of RAD51 are properly regulated. Thus, RADX is essential to achieve the proper balance of RAD51 activity to maintain genome stability.

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