Rev1 plays central roles in mammalian DNA-damage tolerance in response to UV irradiation.

Rev1, a Y-family DNA polymerase, is involved in the tolerance of DNA damage by translesion DNA synthesis (TLS). Previous studies have shown that the C-terminal domain (CTD) and ubiquitin (Ub)-binding (UBM) domains of Rev1 play important roles in UV-damage tolerance, but how these domains contribute to the process remains unclear. In this study, we created Ub mutations in a proliferating cell nuclear antigen (PCNA)-Ub fusion that differentially affect its interaction with Rev1 and Polη and found that UV-damage tolerance depends on its interaction with Rev1 but not Polη. We also created Rev1-UBM mutations altering its interaction with a PCNA-Ub fusion and Rev1-CTD mutations affecting its interaction with Polη and the Rev7 subunit of Polζ. We thus demonstrated that elevated expression of Rev1 alone is sufficient to confer enhanced UV-damage tolerance and that this tolerance depends on its physical interaction with monoubiquitinated PCNA and Polζ but is independent of Polη. Collectively, these studies reveal central roles played by Rev1 in coordinating UV-damage response pathway choice in mammalian cells.

Linear ubiquitin chain induces apoptosis and inhibits tumor growth.

Ubiquitination of proliferating cell nuclear antigen (PCNA) plays an important role in DNA damage response. Ectopic expression of PCNA fused at either terminus with ubiquitin (Ub) lacking two C-terminal glycine residues induces translesion DNA synthesis which resembles synthesis mediated by PCNA monoubiquitination. PCNA fused with Ub containing the C-terminal Gly residues at the C-terminus can be further polyubiquitinated in a Gly-dependent manner, which inhibits cell proliferation and induces ATR-dependent replication checkpoint. In this study, we surprisingly found that PCNA fused to a head-to-tail linear Ub chain induces apoptosis in a Ub chain length-dependent manner. Further investigation revealed that the apoptotic effect is actually induced by the linear Ub chain independently from PCNA, as the Ub chain fused to GFP or an epitope tag still efficiently induces apoptosis. It is revealed that the artificial linear Ub chain differs from endogenously encoded linear Ub chains in that its Ubs contain a Ub-G76S substitution, making the Ub chain resistant to cleavage by deubiquitination enzymes. We demonstrated in this study that ectopic expression of the artificial Ub chain alone in cultured human cancer cells is sufficient to inhibit tumor growth in a xenograft mouse model, making the linear Ub chain a putative anti-cancer agent.

Rev7, the regulatory subunit of Polζ, undergoes UV-induced and Cul4-dependent degradation.

In eukaryotic cells, Rev7 interacts with Rev3 and functions as a regulatory subunit of Polζ, a translesion DNA synthesis (TLS) polymerase. In addition to its role in TLS, mammalian Rev7, also known as Mad2B/Mad2L2, participates in multiple cellular activities including cell cycle progression and double-strand break repair through its interaction with several proteins. Here we show that in mammalian cells, Rev7 undergoes ubiquitin/proteasome-mediated degradation upon UV irradiation in a time-dependent manner. We identified the Rev7 N-terminal destruction box as the degron and Cul4A/B as putative E3 ligases in this process. We also show that the nucleotide excision repair (NER) pathway protein HR23B physically interacts and colocalizes with Rev7 in the nuclear foci after UV irradiation and protects Rev7 from accelerated degradation. Furthermore, a similar Rev7 degradation profile was observed in cells treated with the UV-mimetic agent 4-nitroquinoline 1-oxide but not with cisplatin or camptothecin, suggesting a role of the NER pathway protein(s) in UV-induced Rev7 degradation. These data and the observation that cells deficient in Rev7 are sensitized to UV irradiation while excessive Rev7 protects cells from UV-induced DNA damage provide a new insight into the potential interplay between TLS and NER.

PCNA-Ub polyubiquitination inhibits cell proliferation and induces cell-cycle checkpoints.

In response to replication-blocking lesions, proliferating cell nuclear antigen (PCNA) can be sequentially ubiquitinated at the K164 residue leading to 2 modes of DNA-damage tolerance, namely translesion DNA synthesis (TLS) and error-free lesion bypass. Ectopic expression of PCNA fused with ubiquitin (Ub) lacking the 2 C-terminal Gly residues resembles PCNA monoubiquitination-mediated TLS. However, if the fused Ub contains C-terminal Gly residues, it is further polyubiquitinated and inhibits cell proliferation. Unexpectedly, the polyubiquitination chain does not require any surface Lys residues and is likely to be head-to-tail linked. Such PCNA polyubiquitination interferes with replication, arrests cells at the S-phase and activates the p53 checkpoint pathway. The above cell-cycle arrest is reversible in an ATR-dependent manner, as simultaneous inhibition of ATR, but not ATM, induces apoptosis. Since ectopic expression of PCNA-Ub also induces double-strand breaks that colocalize with single-stranded DNA, we infer that this non-canonical PCNA poly-Ub chain serves as a signal to activate ATR checkpoint and recruit double-strand-break repair apparatus.

Error-free DNA-damage tolerance in Saccharomyces cerevisiae.

DNA-damage tolerance (DDT) is an important mechanism for living cells to bypass replication blocks on the template strand. In Saccharomyces cerevisiae, DDT is mediated by the RAD6 epistasis group of genes, consisting of two parallel pathways: error-prone translesion DNA synthesis (TLS), and error-free lesion bypass. The two pathways are activated by sequential ubiquitination of PCNA on the Lys164 residue. When a replication fork is stalled at a lesion, PCNA is first monoubiquitinated by Rad6-Rad18, which leads to the TLS pathway. The subsequent ubiquitination by the Mms2-Ubc13-Rad5 complex on the monoubiquitinated PCNA is to form a Lys63-linked polyubiquitin chain that promotes error-free lesion bypass. While the TLS pathway has been extensively characterized, the molecular events leading to error-free lesion bypass by polyubiquitinated PCNA are largely obscure. Furthermore, PCNA can also be sumoylated at the same Lys164 residue, which helps to recruit Srs2, a helicase and anti-recombinase. This review summarizes recent advances in our understanding of error-free DDT and its interplay with Srs2 and homologous recombination.

DNA-damage tolerance mediated by PCNA*Ub fusions in human cells is dependent on Rev1 but not Polη.

In response to replication-blocking lesions, proliferating cell nuclear antigen (PCNA) can be sequentially ubiquitinated at the K164 residue, leading to two modes of DNA-damage tolerance, namely, translesion DNA synthesis (TLS) and error-free lesion bypass. Although the majority of reported data support a model whereby monoubiquitinated PCNA enhances its affinity for TLS polymerases and hence recruits them to the damage sites, this model has also been challenged by several observations. In this study, we expressed the PCNA-164R and ubiquitin (UB) fusion genes in an inducible manner in an attempt to mimic PCNA monoubiquitination in cultured human cells. It was found that expression of both N- and C-terminal PCNA•Ub fusions conferred significant tolerance to ultraviolet (UV)-induced DNA damage. Surprisingly, depletion of Polη, a TLS polymerase dedicated to bypassing UV-induced pyrimidine dimers, did not alter tolerance conferred by PCNA•Ub. In contrast, depletion of Rev1, another TLS polymerase serving as a scaffold for the assembly of the TLS complex, completely abolished PCNA•Ub-mediated damage tolerance. Similar genetic interactions were confirmed when UV-induced monoubiquitination of endogenous PCNA is abolished by RAD18 deletion. Hence, PCNA•Ub fusions bypass the requirement for PCNA monoubiquitination, and UV damage tolerance conferred by these fusions is dependent on Rev1 but independent of Polη.

Rev3, the catalytic subunit of Polζ, is required for maintaining fragile site stability in human cells.

It has been long speculated that mammalian Rev3 plays an important, yet unknown role(s) during mammalian development, as deletion of Rev3 causes embryonic lethality in mice, whereas no other translesion DNA synthesis polymerases studied to date are required for mouse embryo development. Here, we report that both subunits of Polζ (Rev3 and Rev7) show an unexpected increase in expression during G(2)/M phase, but they localize independently in mitotic cells. Experimental depletion of Rev3 results in a significant increase in anaphase bridges, chromosomal breaks/gaps and common fragile site (CFS) expression, whereas Rev7 depletion primarily causes lagging chromosome defect with no sign of CFS expression. The genomic instability induced by Rev3 depletion seems to be related to replication stress, as it is further enhanced on aphidicolin treatment and results in increased metaphase-specific Fanconi anemia complementation group D type 2 (FANCD2) foci formation, as well as FANCD2-positive anaphase bridges. Indeed, a long-term depletion of Rev3 in cultured human cells results in massive genomic instability and severe cell cycle arrest. The aforementioned observations collectively support a notion that Rev3 is required for the efficient replication of CFSs during G(2)/M phase, and that the resulting fragile site instability in Rev3 knockout mice may trigger cell death during embryonic development.

Roles of sequential ubiquitination of PCNA in DNA-damage tolerance.

Living organisms not only repair DNA damage induced by environmental agents and endogenous cellular metabolites, but have also developed mechanisms to survive in the presence of otherwise lethal lesions. DNA-damage tolerance (DDT) is considered such a mechanism that resumes DNA synthesis in the presence of replication-blocking lesions. Recent studies revealed that DDT in budding yeast is achieved through sequential ubiquitination of DNA polymerase processivity factor, proliferating cell nuclear antigen (PCNA). It is generally believed that monoubiquitinated PCNA promotes translesion DNA synthesis, whereas polyubiquitinated PCNA mediates an error-free mode of lesion bypass. This review will discuss how ubiquitinated PCNA modulates different means of lesion bypass.