14-3-3 Proteins regulate exonuclease 1-dependent processing of stalled replication forks

Area of Science:

• Molecular Biology
• Cell Biology
• Genetics

Background:

• Replication fork integrity is vital for genome stability, regulated by checkpoint-mediated phosphorylation.
• 14-3-3 proteins bind phosphorylated proteins and have an unclear role in DNA replication stress.
• Exonuclease 1 (Exo1) processes stalled replication forks in checkpoint-defective cells.

Purpose of the Study:

• To investigate the role of 14-3-3 proteins in DNA replication stress response.
• To identify in vivo interaction partners of Exonuclease 1 (Exo1).
• To elucidate the mechanism by which 14-3-3 proteins affect replication fork stability.

Main Methods:

• Yeast and mammalian cell culture.
• Co-immunoprecipitation to identify protein interactions.
• Electron microscopy to visualize DNA structures.
• DNA two-dimensional gel electrophoresis to analyze replication forks.

Main Results:

• 14-3-3 proteins were identified as in vivo interaction partners of Exo1 in both yeast and mammalian cells.
• Yeast cells lacking 14-3-3 proteins showed defective Mec1-dependent Exo1 hyperphosphorylation.
• These cells accumulated Exo1-dependent single-stranded DNA (ssDNA) gaps at stalled forks, leading to persistent checkpoint activation and recovery defects.
• 14-3-3 proteins were shown to promote replication fork progression under nucleotide-limiting conditions.

Conclusions:

• 14-3-3 proteins are essential regulators of DNA replication stress response.
• They interact with Exo1 to control its phosphorylation status and processing of stalled forks.
• 14-3-3 proteins promote replication fork progression, stability, and restart, thereby maintaining genome stability.