Relevance of simultaneous mono-ubiquitinations of multiple units of PCNA homo-trimers in DNA damage tolerance.
Relevance of simultaneous mono-ubiquitinations of multiple units of PCNA homo-trimers in DNA damage tolerance.
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DOI：
10.1371/journal.pone.0118775
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Masutani C
中科院分区：
文献类型：
--
作者：
Kanao R;Masuda Y;Deguchi S;Yumoto-Sugimoto M;Hanaoka F;Masutani C
DNA损伤耐受（DDT）途径，包括跨损伤合成（TLS）和其他未知的机制，使恢复从复制停滞在DNA损伤。DDT途径受增殖细胞核抗原（PCNA）K164残基的翻译后修饰调节。特别是，泛素连接酶RAD 18的单泛素化对于Polη介导的TLS至关重要。虽然修饰PCNA对DDT途径的重要性是众所周知的，但其同源三聚体形式（其中三个K164残基存在于单环中）的相关性仍有待阐明。在这里，我们表明，多个单位的PCNA同源三聚体同时单泛素化在体外和体内。RAD18催化重组系统中PCNA同源三聚体的多个单元的顺序单泛素化。外源性PCNA与内源性PCNA在WI 38VA 13细胞转化体中形成异源三聚体。当K164R突变的PCNA在这些细胞中表达的水平，耗尽内源性PCNA同源三聚体，PCNA复合物的多种修饰减少，细胞显示缺陷，紫外线照射后的DDT。值得注意的是，突变型PCNA的异位表达增加了Polη有效、Polη缺陷和REV 1缺失细胞的UV敏感性，表明DDT途径的破坏不同于Polη和REV 1介导的途径。这些结果表明，同时修改的多个单位的PCNA同源三聚体是需要在人类细胞中的某些DDT途径。
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.