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Regulation of proliferating cell nuclear antigen ubiquitination in mammalian cells

Atsuko Niimi1, Stephanie Brown, Simone Sabbioneda

  • 1Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton BN1 9RQ, United Kingdom.

Proceedings of the National Academy of Sciences of the United States of America
|October 11, 2008
PubMed

Insights

Monoubiquitination of proliferating cell nuclear antigen (PCNA) is crucial for DNA repair. This process, triggered by specific DNA-damaging agents, facilitates the switch to specialized DNA polymerases for translesion synthesis.

Area of Science:

  • Molecular Biology
  • DNA Repair Mechanisms
  • Cellular Response to DNA Damage

Background:

  • DNA damage triggers cellular responses to maintain genomic integrity.
  • Proliferating cell nuclear antigen (PCNA) plays a key role in DNA replication and repair.
  • Monoubiquitination of PCNA is a critical step in coordinating DNA repair pathways.

Purpose of the Study:

  • To investigate the role of PCNA monoubiquitination in response to various DNA-damaging agents.
  • To elucidate the factors influencing PCNA ubiquitination kinetics and dependency.
  • To understand the implications of impaired PCNA ubiquitination on cellular sensitivity to DNA damage.

Main Methods:

  • Treatment of human cells with diverse DNA-damaging agents (UV light, methyl methanesulfonate, mitomycin C, bleomycin, camptothecin).
  • Assessment of PCNA ubiquitination levels using immunoblotting.
  • Analysis of DNA repair pathway involvement (RPA, ATR).
  • Utilizing photolyase-expressing cells to remove UV lesions and study persistence of PCNA ubiquitination.

Main Results:

  • PCNA monoubiquitination occurs in response to UV light, methyl methanesulfonate, and mitomycin C, but not bleomycin or camptothecin.
  • Cyclobutane pyrimidine dimers are the primary drivers of UV-induced PCNA ubiquitination.
  • Failure to ubiquitinate PCNA leads to increased sensitivity to UV and methyl methanesulfonate.
  • PCNA ubiquitination is dependent on Replication Protein A (RPA) but independent of ATR signaling.
  • Ubiquitinated PCNA persists long after UV damage removal, suggesting a role in post-lesion bypass.

Conclusions:

  • PCNA monoubiquitination is a conserved response to specific DNA replication fork-blocking lesions.
  • The kinetics and dependency of PCNA ubiquitination vary with the type of DNA damage.
  • PCNA ubiquitination is essential for tolerance to certain DNA damaging agents.
  • A model for translesion synthesis operating behind the replication fork is proposed to explain persistent PCNA ubiquitination.

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