Stn1 supports Mec1 function in protecting stalled replication forks from degradation

Each time a cell divides, it must accurately duplicate its DNA. This complex process can be disrupted by stress or DNA damage, compromising the replication fork, the structure where DNA replication takes place. Unprotected forks can collapse, leading to genome instability, a hallmark of cancer and other diseases. Replication stress threatens genome integrity by exposing replication forks to nucleolytic degradation. In both yeast and humans, the checkpoint kinases Mec1 and Rad53 limit deleterious single-stranded DNA (ssDNA), yet the protective mechanisms remain incompletely defined. In this study, we discovered that Stn1, a protein of the CST complex, plays a critical role in protecting stalled replication forks from degradation, thus restraining ssDNA formation under nucleotide depletion, especially when the main DNA damage checkpoint protein Mec1 is not fully functional. A gain-of-function allele (stn1-L60F) suppresses the sensitivity to replication stress of Mec1-deficient cells and reduces ssDNA at stalled replication forks, whereas a loss-of-function truncation (stn1-ΔC) exacerbates both phenotypes. Mechanistically, Stn1 opposes the resection activities of Mre11, Exo1, and Sgs1 by promoting Polα-primase-dependent fill-in and by limiting their association with stalled replication forks, with the latter mechanism predominating in the suppression exerted by Stn1-L60F. Thus, our findings uncover a role for Stn1 in safeguarding genome stability by acting as a backup to checkpoint pathways to control DNA processing at stressed forks.