Stn1 supports Mec1 function in protecting stalled replication forks from degradation.

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. Here, we identify a role for the CST subunit Stn1 in cooperating with Mec1 to restrain ssDNA formation under nucleotide depletion. 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 Stn1L60F. Thus, Stn1 works with the checkpoint to curb nuclease activity at sites of replication stress.