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

Qin Z, Lu M, Xu X, Hanna M, Shiomi N, Xiao W - Nucleic Acids Res. (2013)

Bottom Line: 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.Surprisingly, depletion of Polη, a TLS polymerase dedicated to bypassing UV-induced pyrimidine dimers, did not alter tolerance conferred by PCNA•Ub.Similar genetic interactions were confirmed when UV-induced monoubiquitination of endogenous PCNA is abolished by RAD18 deletion.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Capital Normal University, Beijing 100048, China, Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon S7N 5E5, Canada and Project for Environmental Dynamics and Radiation Effects, Fukushima Project Headquarters, National Institute of Radiological Sciences, Chiba 263-8555, Japan.

ABSTRACT
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.

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Effects of Polη depletion on DNA-damage tolerance provided by PCNA•Ub fusions. (A and B) Efficacy of the two anti-Polη siRNAs assessed by qRT-PCR (A) and western blot (B) in T-Rex-293 cells. The relative Polη level is indicated underneath the Polη image. (C) Sample images of UV-induced RPA nuclear foci. (D) Percentage of cells with RPA foci after 8 J/m2 UV irradiation, with or without Dox induction and siPolη treatment. The experimental timeline is as in Figure 3A. (E–G) Survival assays of T-Rex 293 stable transfectants in response to UV damage. Cells were transfected with siPolη and treated with Dox for 3 days before UV irradiation. Two days after UV irradiation, viable cells were counted as a measure of UV tolerance. (E) PCNA-K164R, (F) Ub-PCNA and (G) PCNA-Ub.
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gkt542-F4: Effects of Polη depletion on DNA-damage tolerance provided by PCNA•Ub fusions. (A and B) Efficacy of the two anti-Polη siRNAs assessed by qRT-PCR (A) and western blot (B) in T-Rex-293 cells. The relative Polη level is indicated underneath the Polη image. (C) Sample images of UV-induced RPA nuclear foci. (D) Percentage of cells with RPA foci after 8 J/m2 UV irradiation, with or without Dox induction and siPolη treatment. The experimental timeline is as in Figure 3A. (E–G) Survival assays of T-Rex 293 stable transfectants in response to UV damage. Cells were transfected with siPolη and treated with Dox for 3 days before UV irradiation. Two days after UV irradiation, viable cells were counted as a measure of UV tolerance. (E) PCNA-K164R, (F) Ub-PCNA and (G) PCNA-Ub.

Mentions: As monoubiquitinated PCNA is thought to mediate TLS through an enhanced physical association with TLS polymerases, an immediate prediction is that inactivation of cognate TLS polymerase(s) will reverse the tolerance effect conferred by the PCNA•Ub fusions. To test this hypothesis, we depleted cellular Polη by using two different siRNAs, both of which effectively reduced cellular Polη RNA (Figure 4A) and protein (Figure 4B) to 10–20% of normal levels. Depletion of Polη led to an enhanced UV sensitivity, regardless of strain background or assays used (Figure 4D–G). Quantitative analysis indicates that depletion of Polη causes an effect similar to that caused by PCNA-K164R expression, and the two treatments appear to be additive (Figure 4C–E). Depletion of Polη in Ub-PCNA and PCNA-Ub transfected cells also increased RPA focus-positive cells, regardless of whether the fusion proteins were produced (Figure 4D and Supplementary Figure S4A). Quantitative analysis indicates that the tolerant effect conferred by PCNA•Ub fusions and the sensitive effect caused by Polη depletion are two independent events, and the net results are the simple sum of two separate treatments. The survival assay results also indicate that the two treatments appear to offset each other so that cells returned to the wild-type state (Figure 4F and G), which reinforces the conclusions drawn from the RPA nuclear focus assay. From the aforementioned analyses, we conclude that the DNA-damage tolerance conferred by PCNA•Ub fusions does not rely on functional Polη.Figure 4.


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

Qin Z, Lu M, Xu X, Hanna M, Shiomi N, Xiao W - Nucleic Acids Res. (2013)

Effects of Polη depletion on DNA-damage tolerance provided by PCNA•Ub fusions. (A and B) Efficacy of the two anti-Polη siRNAs assessed by qRT-PCR (A) and western blot (B) in T-Rex-293 cells. The relative Polη level is indicated underneath the Polη image. (C) Sample images of UV-induced RPA nuclear foci. (D) Percentage of cells with RPA foci after 8 J/m2 UV irradiation, with or without Dox induction and siPolη treatment. The experimental timeline is as in Figure 3A. (E–G) Survival assays of T-Rex 293 stable transfectants in response to UV damage. Cells were transfected with siPolη and treated with Dox for 3 days before UV irradiation. Two days after UV irradiation, viable cells were counted as a measure of UV tolerance. (E) PCNA-K164R, (F) Ub-PCNA and (G) PCNA-Ub.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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gkt542-F4: Effects of Polη depletion on DNA-damage tolerance provided by PCNA•Ub fusions. (A and B) Efficacy of the two anti-Polη siRNAs assessed by qRT-PCR (A) and western blot (B) in T-Rex-293 cells. The relative Polη level is indicated underneath the Polη image. (C) Sample images of UV-induced RPA nuclear foci. (D) Percentage of cells with RPA foci after 8 J/m2 UV irradiation, with or without Dox induction and siPolη treatment. The experimental timeline is as in Figure 3A. (E–G) Survival assays of T-Rex 293 stable transfectants in response to UV damage. Cells were transfected with siPolη and treated with Dox for 3 days before UV irradiation. Two days after UV irradiation, viable cells were counted as a measure of UV tolerance. (E) PCNA-K164R, (F) Ub-PCNA and (G) PCNA-Ub.
Mentions: As monoubiquitinated PCNA is thought to mediate TLS through an enhanced physical association with TLS polymerases, an immediate prediction is that inactivation of cognate TLS polymerase(s) will reverse the tolerance effect conferred by the PCNA•Ub fusions. To test this hypothesis, we depleted cellular Polη by using two different siRNAs, both of which effectively reduced cellular Polη RNA (Figure 4A) and protein (Figure 4B) to 10–20% of normal levels. Depletion of Polη led to an enhanced UV sensitivity, regardless of strain background or assays used (Figure 4D–G). Quantitative analysis indicates that depletion of Polη causes an effect similar to that caused by PCNA-K164R expression, and the two treatments appear to be additive (Figure 4C–E). Depletion of Polη in Ub-PCNA and PCNA-Ub transfected cells also increased RPA focus-positive cells, regardless of whether the fusion proteins were produced (Figure 4D and Supplementary Figure S4A). Quantitative analysis indicates that the tolerant effect conferred by PCNA•Ub fusions and the sensitive effect caused by Polη depletion are two independent events, and the net results are the simple sum of two separate treatments. The survival assay results also indicate that the two treatments appear to offset each other so that cells returned to the wild-type state (Figure 4F and G), which reinforces the conclusions drawn from the RPA nuclear focus assay. From the aforementioned analyses, we conclude that the DNA-damage tolerance conferred by PCNA•Ub fusions does not rely on functional Polη.Figure 4.

Bottom Line: 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.Surprisingly, depletion of Polη, a TLS polymerase dedicated to bypassing UV-induced pyrimidine dimers, did not alter tolerance conferred by PCNA•Ub.Similar genetic interactions were confirmed when UV-induced monoubiquitination of endogenous PCNA is abolished by RAD18 deletion.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Capital Normal University, Beijing 100048, China, Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon S7N 5E5, Canada and Project for Environmental Dynamics and Radiation Effects, Fukushima Project Headquarters, National Institute of Radiological Sciences, Chiba 263-8555, Japan.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus