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Genetic Interactions Implicating Postreplicative Repair in Okazaki Fragment Processing

Jordan R Becker1, Carles Pons2, Hai Dang Nguyen1

  • 1Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, United States of America.

Plos Genetics
|November 7, 2015
PubMed

Insights

Ubiquitination of proliferating cell nuclear antigen (PCNA) at K164 repairs DNA replication gaps. This study shows PCNA ubiquitination also responds to lagging strand replication defects, extending its role in DNA repair pathways.

Area of Science:

  • Molecular Biology
  • DNA Replication
  • Cellular Stress Response

Background:

  • Proliferating cell nuclear antigen (PCNA) ubiquitination at K164 is crucial for postreplicative repair (PRR) of DNA gaps caused by stalled polymerases.
  • The role of PCNA ubiquitination in response to replication defects not directly impairing DNA synthesis remained unclear.

Purpose of the Study:

  • To investigate whether cells utilize PRR in response to replication defects unrelated to direct DNA synthesis impairment.
  • To explore the connection between PCNA ubiquitination and lagging strand replication fidelity.

Main Methods:

  • Synthetic genetic array (SGA) analysis was employed using a ubiquitination-deficient PCNA mutant (K164R) in Saccharomyces cerevisiae.
  • Genetic interactions and cell viability were assessed in conjunction with mutations affecting lagging strand replication (e.g., rad27Δ, elg1Δ).
  • PCNA ubiquitination levels and S phase checkpoint activation (Rad53 phosphorylation) were monitored under various conditions.

Main Results:

  • SGA analysis revealed a significant correlation between the PCNA K164R mutant and mutants deficient in lagging strand replication processes.
  • Chronic PCNA ubiquitination at K164 was observed in rad27Δ and elg1Δ mutants, indicating its involvement in processing errors.
  • PCNA ubiquitination was found to suppress replication stress from defective Okazaki fragment maturation, and its ablation enhanced S phase checkpoint activation.
  • Overexpression of exonuclease 1 reduced PCNA ubiquitination, suggesting unprocessed flaps may trigger PRR signaling.

Conclusions:

  • PCNA ubiquitination at K164 is a critical response not only to DNA synthesis stalling but also to defects in lagging strand DNA processing.
  • Unprocessed flaps during Okazaki fragment maturation can signal for PRR activation via PCNA ubiquitination.
  • This extends the known functions of PCNA ubiquitination in maintaining genome stability during DNA replication.

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