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UBR5 interacts with the replication fork and protects DNA replication from DNA polymerase η toxicity

Lina Cipolla1, Federica Bertoletti1, Antonio Maffia1

  • 1Istituto di Genetica Molecolare "Luigi Luca Cavalli-Sforza", CNR, Pavia, Italy.

Nucleic Acids Research
|October 6, 2019
PubMed

Insights

The E3 ligase UBR5 is crucial for DNA replication, and its depletion causes problems. Blocking histone H2A ubiquitylation rescues cells, highlighting its role in translesion synthesis (TLS) regulation.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Genetics

Background:

  • Accurate DNA replication is essential for genome stability and cell survival.
  • Cancer often involves disruptions in DNA replication and damage signaling pathways.
  • Post-translational modifications, particularly ubiquitylation, are key regulators of DNA replication and repair.

Purpose of the Study:

  • To investigate the role of the E3 ligase UBR5 in DNA replication.
  • To elucidate the connection between UBR5, histone H2A ubiquitylation (UbiH2A), and translesion synthesis (TLS) polymerase polη.
  • To understand how UBR5 depletion impacts replication fork progression and genome integrity.

Main Methods:

  • Co-immunoprecipitation to identify UBR5 interacting partners.
  • RNA interference (RNAi) to deplete UBR5 expression.
  • Flow cytometry to analyze S-phase progression and cell cycle.
  • Western blotting to assess protein levels and modifications, including UbiH2A.
  • Analysis of single-stranded DNA accumulation.

Main Results:

  • UBR5 interacts with replication fork components, including TLS polymerase polη.
  • Depletion of UBR5 impairs DNA replication, leading to slower S-phase progression and single-stranded DNA accumulation.
  • UBR5 knockdown effects are linked to dysregulation of histone H2A ubiquitylation (UbiH2A).
  • Inhibition of UbiH2A is sufficient to rescue cells from UBR5 depletion-induced replication defects.
  • The presence of polη is identified as the primary driver of replication defects and cell death upon UBR5 silencing.
  • A novel interaction between polη and histone H2A is discovered.

Conclusions:

  • UBR5 plays a critical role in maintaining DNA replication fidelity.
  • Histone H2A ubiquitylation (UbiH2A) is a key regulatory mechanism in response to UBR5 depletion.
  • Polη is a major contributor to replication stress phenotypes observed in UBR5-depleted cells.
  • The interaction between polη and H2A suggests a novel pathway for regulating TLS recruitment and activity via UbiH2A.

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