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

Rie Kanao; Yuji Masuda; Saori Deguchi; Mayumi Yumoto-Sugimoto; Fumio Hanaoka; Chikahide Masutani

doi:10.1371/journal.pone.0118775

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.

Authors

Rie Kanao¹, Yuji Masuda², Saori Deguchi³, Mayumi Yumoto-Sugimoto³, Fumio Hanaoka⁴, Chikahide Masutani¹

Affiliations

¹ Department of Genome Dynamics, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan; Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.
² Department of Genome Dynamics, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan; Department of Toxicogenomics, Nagoya University Graduate School of Medicine, Nagoya, Japan.
³ Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.
⁴ Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan; Faculty of Science, Gakushuin University, Tokyo, Japan.

Abstract

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

Substances

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.