Relevance of simultaneous mono-ubiquitinations of multiple units of PCNA homo-trimers in DNA damage tolerance

PLoS One. 2015 Feb 18;10(2):e0118775. doi: 10.1371/journal.pone.0118775. eCollection 2015.


DNA damage tolerance (DDT) pathways, including translesion synthesis (TLS) and additional unknown mechanisms, enable recovery from replication arrest at DNA lesions. DDT pathways are regulated by post-translational modifications of proliferating cell nuclear antigen (PCNA) at its K164 residue. In particular, mono-ubiquitination by the ubiquitin ligase RAD18 is crucial for Polη-mediated TLS. Although the importance of modifications of PCNA to DDT pathways is well known, the relevance of its homo-trimer form, in which three K164 residues are present in a single ring, remains to be elucidated. Here, we show that multiple units of a PCNA homo-trimer are simultaneously mono-ubiquitinated in vitro and in vivo. RAD18 catalyzed sequential mono-ubiquitinations of multiple units of a PCNA homo-trimer in a reconstituted system. Exogenous PCNA formed hetero-trimers with endogenous PCNA in WI38VA13 cell transformants. When K164R-mutated PCNA was expressed in these cells at levels that depleted endogenous PCNA homo-trimers, multiple modifications of PCNA complexes were reduced and the cells showed defects in DDT after UV irradiation. Notably, ectopic expression of mutant PCNA increased the UV sensitivities of Polη-proficient, Polη-deficient, and REV1-depleted cells, suggesting the disruption of a DDT pathway distinct from the Polη- and REV1-mediated pathways. These results suggest that simultaneous modifications of multiple units of a PCNA homo-trimer are required for a certain DDT pathway in human cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • DNA Damage
  • DNA Repair / radiation effects
  • DNA-Binding Proteins / metabolism*
  • DNA-Directed DNA Polymerase / metabolism
  • Humans
  • In Vitro Techniques
  • Lysine / genetics*
  • Male
  • Mutation
  • Proliferating Cell Nuclear Antigen / chemistry*
  • Proliferating Cell Nuclear Antigen / genetics*
  • Proliferating Cell Nuclear Antigen / metabolism
  • Ubiquitin-Protein Ligases
  • Ubiquitination


  • DNA-Binding Proteins
  • Proliferating Cell Nuclear Antigen
  • RAD18 protein, human
  • Ubiquitin-Protein Ligases
  • DNA-Directed DNA Polymerase
  • Lysine

Grants and funding

This work was supported by KAKENHI [26740017 to RK, 24310040 and 26550024 to YM, 22131008 to FH, and 25241011 to CM] from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by the Mitsubishi Foundation, the Takeda Science Foundation, and the Uehara Memorial Foundation [to CM]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.