Checkpoint-mediated DNA polymerase e 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 in balance, due to partitioning of nascent strands between catalytic sites, so that net end resection occurs when synthesis is impaired. Stalling of chromosomal DNA synthesis activates replication checkpoint kinases, required to preserve the functional integrity of replication forks. We found that Pole is phosphorylated in a Rad53CHK1-dependent manner upon fork stalling, likely to limit Pole-driven nascent strand resection that causes replication fork collapse. In stress conditions Pole phosphorylation occurs on serine 430 of the Pol2 catalytic subunit. A S430 phosphomimic limits strand partitioning and exonucleolytic processivity, while non-phosphorylatable Pol2-S430A bypasses checkpoint regulation causing stalled fork resection and collapse. We propose that checkpoint kinases switch Pole to an exonuclease-safe mode by curbing active site partitioning thus preventing nascent strand resection and stabilizing stalled replication forks. Comparative Genome Sequencing (CGS) was carried out as decribed (Frattini et al., Mol Cell 2017). In brief, genomic DNA was extracted from cells arrested in G1 or after different times following release into S-phase in the presence of hydroxyurea (HU). Genomic DNA was sequenced, reads were mapped to the reference genome for each sample and S/G1 read number ratios were used to plot replication profiles along the S.cerevisiae genome.