Stn1 supports Mec1 function in protecting stalled replication forks from degradation

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• Replication stress poses a significant threat to genome integrity.
• Checkpoint kinases like Mec1 and Rad53 are crucial for limiting DNA damage.
• The precise mechanisms by which cells protect against replication stress are not fully understood.

Purpose of the Study:

• To investigate the role of the CST subunit Stn1 in genome protection during replication stress.
• To elucidate how Stn1 cooperates with Mec1 to limit single-stranded DNA (ssDNA) formation.
• To understand the molecular mechanisms underlying Stn1's protective function.

Main Methods:

• Utilized yeast genetics, including gain-of-function (stn1-L60F) and loss-of-function (stn1-ΔC) alleles.
• Assessed sensitivity to replication stress in Mec1-deficient cells.
• Quantified ssDNA accumulation at stalled replication forks.
• Investigated the interaction of Stn1 with nucleases (Mre11, Exo1, Sgs1) and Polα-primase.

Main Results:

• A gain-of-function mutation in Stn1 (stn1-L60F) suppressed replication stress sensitivity in Mec1-deficient cells.
• Stn1 deficiency (stn1-ΔC) exacerbated replication stress sensitivity and ssDNA accumulation.
• Stn1 limits ssDNA by promoting Polα-primase-dependent fill-in and by restricting nuclease association with stalled forks.
• The suppression by stn1-L60F primarily involves limiting nuclease association with replication forks.

Conclusions:

• Stn1 plays a critical role in cooperating with the Mec1 checkpoint pathway to maintain genome integrity under replication stress.
• Stn1 acts as a safeguard by inhibiting the activity of resection nucleases at stalled replication forks.
• These findings reveal a novel mechanism by which Stn1 contributes to genome stability.