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

Erika Casari1, Flavio Corallo1, Luca Edoardo Milani1

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Summary

The CST subunit Stn1 collaborates with Mec1 to prevent genome damage during replication stress. Stn1 limits single-stranded DNA formation by inhibiting nucleases, thus protecting stalled replication forks.

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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.