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.
Highlights
The stability of genomic DNA is challenged by various DNA-damaging agents
It is likely that the proliferating cell nuclear antigen (PCNA)[KR] homo-trimers competed with the PCNA-WT homo-trimers for loading onto DNA, thereby impeding mono-ubiquitination of the WT molecules by RAD18
This study sheds light on the role of multiple mono-ubiquitinations of PCNA homo-trimers in the regulation of DNA damage tolerance (DDT) pathways and unveils the regulatory mechanism of a minor DDT pathway that is masked by polymerase η (Polη)-mediated Translesion synthesis (TLS), the prominent DDT pathway, in human cells
Summary
The stability of genomic DNA is challenged by various DNA-damaging agents. Replication fork progression is disrupted by DNA lesions; cells have evolved DNA damage tolerance (DDT) pathways that rescue stalled replication forks. PCNA forms a ring-shaped homo-trimer complex that interacts with many proteins and plays crucial roles in DNA replication, DNA repair, and cell-cycle control [6]. It has been reported that Ub-PCNA promotes Polη-dependent TLS and a Polη-independent DDT pathway [9, 10], and multiple two TLS polymerases mechanisms have been proposed [11, 12]. The importance of post-translational modifications of PCNA to the control of DDT pathways is well known, the relevance of its homo-trimer form has not been examined in detail. Experiments using human cells ectopically expressing wild-type (WT) or K164-mutated PCNA revealed that mutation of this lysine residue disrupts mono-ubiquitination of the multiple units within a homo-trimer complex as well as a DDT pathway that is distinct from Polη-mediated TLS
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