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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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Related in: MedlinePlus

Phenotypic evaluation of in vivo PCNA•Ub fusions. (A) A schematic timeline for the RPA focus assay. (B–D) Percentage of RPA foci positive cells after UV irradiation. (E) A schematic timeline for the cell survival assay. (F–H) Relative sensitivity of T-Rex-293 transfectants to UV-induced growth inhibition. (B and F) The PCNA-K164R transfectants. (C and G) The Ub-PCNA transfectants. (D and H) The PCNA-Ub transfectants. The results are the average of at least three independent experiments with standard deviations.
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gkt542-F3: Phenotypic evaluation of in vivo PCNA•Ub fusions. (A) A schematic timeline for the RPA focus assay. (B–D) Percentage of RPA foci positive cells after UV irradiation. (E) A schematic timeline for the cell survival assay. (F–H) Relative sensitivity of T-Rex-293 transfectants to UV-induced growth inhibition. (B and F) The PCNA-K164R transfectants. (C and G) The Ub-PCNA transfectants. (D and H) The PCNA-Ub transfectants. The results are the average of at least three independent experiments with standard deviations.

Mentions: Compared with non-induced cells, expression of PCNA-K164R or PCNA•Ub fusions alone did not affect cell cycle progression (Supplementary Figure S2) or proliferation (Figure 3F–H) in the absence of DNA damage, indicating that PCNA•Ub fusion proteins do not affect normal DNA replication. The aforementioned observations set a desired platform for the investigation of PCNA•Ub fusion functions.Figure 3.


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)

Phenotypic evaluation of in vivo PCNA•Ub fusions. (A) A schematic timeline for the RPA focus assay. (B–D) Percentage of RPA foci positive cells after UV irradiation. (E) A schematic timeline for the cell survival assay. (F–H) Relative sensitivity of T-Rex-293 transfectants to UV-induced growth inhibition. (B and F) The PCNA-K164R transfectants. (C and G) The Ub-PCNA transfectants. (D and H) The PCNA-Ub transfectants. The results are the average of at least three independent experiments with standard deviations.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3753651&req=5

gkt542-F3: Phenotypic evaluation of in vivo PCNA•Ub fusions. (A) A schematic timeline for the RPA focus assay. (B–D) Percentage of RPA foci positive cells after UV irradiation. (E) A schematic timeline for the cell survival assay. (F–H) Relative sensitivity of T-Rex-293 transfectants to UV-induced growth inhibition. (B and F) The PCNA-K164R transfectants. (C and G) The Ub-PCNA transfectants. (D and H) The PCNA-Ub transfectants. The results are the average of at least three independent experiments with standard deviations.
Mentions: Compared with non-induced cells, expression of PCNA-K164R or PCNA•Ub fusions alone did not affect cell cycle progression (Supplementary Figure S2) or proliferation (Figure 3F–H) in the absence of DNA damage, indicating that PCNA•Ub fusion proteins do not affect normal DNA replication. The aforementioned observations set a desired platform for the investigation of PCNA•Ub fusion functions.Figure 3.

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