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Exo1 phosphorylation inhibits exonuclease activity and prevents fork collapse in rad53 mutants independently of the

Esther C Morafraile1, Alberto Bugallo1, Raquel Carreira2

  • 1Instituto de Biología Funcional y Genómica (CSIC/USAL), Campus Miguel de Unamuno, Salamanca 37007, Spain.

Nucleic Acids Research
|February 6, 2020
PubMed

Insights

Rad53-dependent phosphorylation of Exo1 is crucial for stabilizing stalled replication forks during S phase. This phosphorylation inhibits Exo1

Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • The S phase checkpoint is essential for maintaining genome stability during DNA replication.
  • Exo1 is a nuclease involved in DNA repair and replication fork stability, and its phosphorylation is observed under genotoxic stress.
  • The precise role of checkpoint-dependent Exo1 phosphorylation in regulating its activity and preserving fork stability remains unclear.

Purpose of the Study:

  • To elucidate the role of Rad53-dependent phosphorylation in controlling Exo1 activity and maintaining replication fork stability.
  • To determine whether Exo1's 5' to 3' exonuclease or flap endonuclease activity is responsible for fork collapse in checkpoint mutants.

Main Methods:

  • Investigated Exo1 phosphorylation in response to DNA damage and dNTP depletion.
  • Generated phospho-mimic exo1 alleles to assess their effect on fork stability in rad53 mutants.
  • Assessed the role of Bmh1-2 (14-3-3 proteins) in Exo1-mediated fork stabilization.

Main Results:

  • Exo1 phosphorylation is dependent on Rad53, not Dun1, in response to genotoxic stress or dNTP depletion.
  • Phospho-mimic exo1 alleles effectively rescued fork collapse in rad53 mutants, similar to nuclease-dead exo1 alleles.
  • This rescue was independent of Bmh1-2, indicating 14-3-3 proteins are dispensable for fork stabilization when Exo1 is downregulated.
  • Phosphorylation specifically inhibits Exo1's 5' to 3' exonuclease activity.

Conclusions:

  • Rad53-dependent phosphorylation of Exo1 is the primary mechanism for preserving replication fork stability under replicative stress.
  • The 5' to 3' exonuclease activity of Exo1, not its flap endonuclease activity, is responsible for replication fork collapse in checkpoint-deficient mutants.
  • 14-3-3 proteins are not essential for Exo1-mediated fork stabilization.

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