title: Relevance of simultaneous mono-ubiquitinations of multiple units of PCNA homo-trimers in DNA damage tolerance.
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.
authors.affiliation: Department of Genome Dynamics, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan; Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.
authors.forename: Rie
authors.initials: R
authors.lastname: Kanao
authors.affiliation: Department of Genome Dynamics, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan; Department of Toxicogenomics, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Masuda
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.
authors.forename: Saori
authors.initials: S
authors.lastname: Deguchi
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.
authors.forename: Mayumi
authors.initials: M
authors.lastname: Yumoto-Sugimoto
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan; Faculty of Science, Gakushuin University, Tokyo, Japan.
authors.forename: Fumio
authors.initials: F
authors.lastname: Hanaoka
authors.affiliation: Department of Genome Dynamics, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan; Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.
authors.forename: Chikahide
authors.initials: C
authors.lastname: Masutani
mesh_terms: D002460:Cell Line; D004249:DNA Damage; D004260:DNA Repair / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008239:Lysine / Q000235:genetics*; D008297:Male; D009154:Mutation; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases; D054875:Ubiquitination
chemical_list: D004268:DNA-Binding Proteins; D018809:Proliferating Cell Nuclear Antigen; C485954:RAD18 protein, human; D044767:Ubiquitin-Protein Ligases; D004259:DNA-Directed DNA Polymerase; D008239:Lysine