S-phase checkpoint protects from aberrant replication fork processing and degradation.

Replication stress, a hallmark of cancer cells, is detected by checkpoint mechanisms that trigger a range of cellular responses. Among these, the preservation of replication fork integrity is crucial for ensuring survival in the presence of DNA damage. In budding yeast checkpoint mutants, DNA damage leads to irreversible replication fork arrest and subsequent cell death, though the underlying mechanism remains unclear. Our study reveals that several DNA processing factors, including Rad51, the Rad5 HIRAN and helicase domains, and the catalytic activity of Mus81, contribute to this lethality. Nevertheless, their roles are masked by their essential functions in cell survival after damage removal. Additionally, we show that these factors, along with Exo1, drive the gradual degradation of nascent DNA at replication forks upon DNA damage. Notably, this degradation can be mitigated by expression of human PrimPol, which is absent in yeast. These findings suggest that the essential role of S-phase checkpoints upon DNA damage is to safeguard stalled replication forks from aberrant processing, thereby preserving nascent DNA integrity.