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DNA replication is initiated at sites containing predefined DNA sequences known as origins of replication. DNA is unwound at these sites by the minichromosome maintenance (MCM) helicase and other factors such as Cdc45 and the associated GINS complex.The unwound single strands are protected by replication protein A (RPA) until DNA polymerase starts synthesizing DNA at the 5’ end of the strand in the same direction as the replication fork. To prevent the replication fork from falling apart,...
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S-phase checkpoint protects from aberrant replication fork processing and degradation.

Iván Núñez-Martín1,2, Lucy S Drury3, María I Martínez-Jiménez4

  • 1Andalusian Center of Molecular Biology and Regenerative Medicine, Universidad de Sevilla-CSIC-Universidad Pablo de Olavide, Seville 41092, Spain.

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|July 30, 2025
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Summary
This summary is machine-generated.

Replication stress in cancer cells is managed by checkpoints. Our study shows DNA processing factors cause cell death by degrading nascent DNA at stalled forks, a process preventable by human PrimPol.

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Area of Science:

  • Molecular Biology
  • Cell Biology
  • Genetics

Background:

  • Replication stress is a common feature in cancer cells, necessitating robust checkpoint mechanisms to maintain genomic stability.
  • The integrity of replication forks during DNA damage is critical for cell survival, yet the precise mechanisms leading to cell death in checkpoint mutants remain elusive.

Purpose of the Study:

  • To elucidate the molecular mechanisms underlying replication fork instability and cell death in budding yeast checkpoint mutants exposed to DNA damage.
  • To identify the specific DNA processing factors involved in the aberrant processing of stalled replication forks.

Main Methods:

  • Utilized budding yeast (Saccharomyces cerevisiae) as a model organism.
  • Employed genetic analysis of checkpoint mutants and DNA processing factors (Rad51, Rad5, Mus81, Exo1).
  • Assessed replication fork integrity and nascent DNA degradation through molecular assays, including the impact of human PrimPol expression.

Main Results:

  • Identified Rad51, Rad5 (HIRAN and helicase domains), and Mus81 catalytic activity as contributors to cell death in checkpoint mutants.
  • Demonstrated that Exo1, along with the aforementioned factors, drives the degradation of nascent DNA at stalled replication forks upon DNA damage.
  • Showed that expressing human PrimPol in yeast mitigates this nascent DNA degradation.

Conclusions:

  • S-phase checkpoints are essential for preventing the detrimental processing of stalled replication forks during DNA damage.
  • Aberrant processing by DNA processing factors leads to nascent DNA degradation and cell death in the absence of functional checkpoints.
  • Safeguarding nascent DNA integrity at stalled replication forks is a critical function of S-phase checkpoints.