Stn1 supports Mec1 function in protecting stalled replication forks from degradation

Replication stress threatens genome stability by exposing stalled replication forks to nucleolytic degradation and excessive single-stranded DNA accumulation (ssDNA). Although the checkpoint kinases Mec1 and Rad53 protect stalled forks, the mechanisms limiting aberrant DNA processing remain unclear. We identify the CST subunit Stn1 as a key factor cooperating with Mec1 to preserve replication fork integrity under replication stress. A gain-of-function Stn1 allele suppresses the hypersensitivity of Mec1-deficient cells and reduces ssDNA accumulation at stalled forks, whereas Stn1 loss of function exacerbates fork degradation. Mechanistically, Stn1 counteracts Mre11-, Exo1- and Sgs1-mediated resection by promoting Polα–primase-dependent fill-in synthesis and, predominantly, by restricting nuclease association with stalled forks. Stn1 also limits nuclease recruitment at DNA double-strand breaks, revealing a broader role in regulating DNA end processing and maintaining genome stability.