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

## TL;DR

This study shows that the protein Stn1 helps protect DNA replication forks from damage, especially when another key protein, Mec1, is not working properly.

## Contribution

The study reveals a novel role for Stn1 in cooperating with Mec1 to prevent DNA degradation at stalled replication forks.

## Key findings

- Stn1 limits nucleases from resecting stalled replication forks, reducing ssDNA formation.
- A gain-of-function mutation in Stn1 (L60F) enhances protection against replication stress in Mec1-deficient cells.
- Stn1 primarily suppresses nuclease activity by limiting their association with stalled forks.

## Abstract

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.

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. In this study, we discovered that Stn1, a protein of the CST complex, plays a critical role in protecting stalled replication forks from degradation, especially when the main DNA damage checkpoint protein Mec1 is not fully functional. Stn1 prevents the accumulation of single-stranded DNA (ssDNA) by limiting nucleases, the enzymes that degrade DNA, from excessively resecting replication forks. Remarkably, we identified a specific mutation in Stn1 (L60F) that enhances this protective function. 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.