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The DNA replication checkpoint limits Okazaki fragment accumulation to protect and restart stalled forks.

Berta Canal·Agostina P Bertolin·Giselle C Lee·Lucy S Drury·Masashi Minamino·John F X Diffley
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Abstract

Understanding how DNA replication forks stall and restart and how the DNA replication checkpoint prevents irreversible fork collapse in molecular detail are crucial for understanding how cells maintain stable genomes and how they prevent the genetic instability that drives cancer. Here, we describe the reconstitution of fork stalling and restart with purified budding yeast proteins. After nucleotide depletion, leading-strand DNA synthesis quickly stops but CMG helicase continues to unwind, and Okazaki fragments continue to initiate on the lagging strand. Incomplete Okazaki fragments sequester PCNA, RFC, and DNA polymerases δ and ε, which prevents normal DNA synthesis restart and exposes nascent DNA to nuclease attack. The DNA replication checkpoint restrains fork progression, which limits this sequestration, protecting stalled forks from collapse and ensuring restart.

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DNA ReplicationSaccharomyces cerevisiaeSaccharomyces cerevisiae ProteinsProliferating Cell Nuclear AntigenDNA HelicasesDNA, FungalDNA Polymerase IIIDNA Polymerase IIDNAReplication Protein CGenomic InstabilityDNA polymerase αDNA polymerase δDNA polymerase εDNA replication checkpointDNA replication fork stabilizationOkazaki fragmentsPCNARFCRPA exhaustionreplication fork collapseJournal Article

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Berta Canal, Agostina P Bertolin, Giselle C Lee, Lucy S Drury, Masashi Minamino, John F X Diffley. 2025-06-26. The DNA replication checkpoint limits Okazaki fragment accumulation to protect and restart stalled forks.. https://doi.org/10.1016/j.molcel.2025.06.001

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