Stn1 supports Mec1 function in protecting stalled replication forks from degradation

Replication stress poses a significant threat to genome integrity by increasing the vulnerability of replication forks to nucleolytic degradation. In both yeast and humans, the checkpoint kinases Mec1 and Rad53 prevent the generation of deleterious single-stranded DNA through a process that remains poorly understood. Here, we show that the Stn1 subunit of the CST complex cooperates with the checkpoint kinase Mec1 to limit ssDNA generation under replication stress. A Stn1 mutation suppresses the sensitivity to replication stress of Mec1- defective yeast cells and reduces ssDNA levels at stalled forks, whereas both sensitivity and ssDNA accumulation are exacerbated by Stn1 loss of function. Mechanistically, Stn1 counteracts the resection activities of Mre11, Exo1, and Sgs1 by limiting their association not only at stalled replication forks but also at DNA double-strand breaks, revealing that Stn1 functions alongside checkpoint pathways in protecting DNA from nuclease-mediated degradation.