Checkpoint-Mediated DNA Polymerase ε Exonuclease Activity Curbing Counteracts Resection-Driven Fork Collapse

DNA polymerase epsilon (Pole) carries out leading strand synthesis with high fidelity owing to its exonuclease activity. Pole polymerase and exonuclease activities are balanced, due to partitioning of nascent DNA strands between catalytic sites, so that net resection occurs when synthesis is impaired. In vivo, DNA synthesis stalling activates replication checkpoint kinases, which act to preserve the functional integrity of replication forks. Here we show that stalled Pole drives nascent strand resection causing fork functional collapse, averted via checkpoint-dependent phosphorylation. Pole catalytic subunit Pol2 is phosphorylated on serine 430, which influences partitioning between polymerase and exonuclease active sites. A phosphormimetic S430D change reduces exonucleolysis in vitro and counteracts fork functional collapse. Conversely, non-phosphorylatable pol2-S430A expression results in resection-driven collapse of stressed forks. Our findings reveal that checkpoint kinases switch Pole to an exonuclease-safe mode preventing nascent strand resection and stabilizing stalled replication forks. Elective partitioning suppression has implications for the diverse Pole roles in genome integrity maintenance.