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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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Establishment of stable cell lines expressing different forms of PCNA. (A) Schematic diagrams of PCNA•Ub fusion constructs. All ORFs contain a K164R mutation to prevent in vivo PCNA ubiquitination. In addition, the Ub-coding region is either fused to PCNA at the N-terminus (Ub-PCNA) or at the C-terminus (PCNA-Ub). All three ORFs are cloned into plasmid pcDNA5.0FRT/TO. (B) Ectopic expression of different forms of PCNA in stably transfected T-Rex-293 cells. Dox-induced expression of ectopic PCNA was analyzed by western blot using an anti-PCNA antibody, and the cellular β-actin serves as a loading control. PCNA-Ub and Ub-PCNA migrate differently, although they have the same molecular weight. (C) Polη and Rev1 preferentially bind to PCNA-Ub over PCNA. T-Rex-293/PCNA-Ub cells were transfected with GFP-Polη or GFP-mRev1 and incubated for 6 h followed by Dox treatment for 2 days. Immunoprecipitation was performed by GFP-Trap-A beads, and the products were analyzed by western blot. (D) The Ub-I44 residue is required for the enhanced affinity between PCNA-Ub and GFP-Polη. T-Rex-293/PCNA-Ub and T-Rex-293/PCNA-Ub-I44A cells were transfected with GFP-Polη, incubated for 6 h followed by Dox treatment for 2 days. Immunoprecipitation was performed by GFP-Trap-A beads, and the products were analyzed by western blot.
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gkt542-F1: Establishment of stable cell lines expressing different forms of PCNA. (A) Schematic diagrams of PCNA•Ub fusion constructs. All ORFs contain a K164R mutation to prevent in vivo PCNA ubiquitination. In addition, the Ub-coding region is either fused to PCNA at the N-terminus (Ub-PCNA) or at the C-terminus (PCNA-Ub). All three ORFs are cloned into plasmid pcDNA5.0FRT/TO. (B) Ectopic expression of different forms of PCNA in stably transfected T-Rex-293 cells. Dox-induced expression of ectopic PCNA was analyzed by western blot using an anti-PCNA antibody, and the cellular β-actin serves as a loading control. PCNA-Ub and Ub-PCNA migrate differently, although they have the same molecular weight. (C) Polη and Rev1 preferentially bind to PCNA-Ub over PCNA. T-Rex-293/PCNA-Ub cells were transfected with GFP-Polη or GFP-mRev1 and incubated for 6 h followed by Dox treatment for 2 days. Immunoprecipitation was performed by GFP-Trap-A beads, and the products were analyzed by western blot. (D) The Ub-I44 residue is required for the enhanced affinity between PCNA-Ub and GFP-Polη. T-Rex-293/PCNA-Ub and T-Rex-293/PCNA-Ub-I44A cells were transfected with GFP-Polη, incubated for 6 h followed by Dox treatment for 2 days. Immunoprecipitation was performed by GFP-Trap-A beads, and the products were analyzed by western blot.

Mentions: Several considerations were taken into account when making the PCNA and Ub fusion (PCNA•Ub) constructs. First, the PCNA ORF used for fusions contains a K164R mutation to prevent in vivo PCNA ubiquitination at the K164 residue following UV irradiation. Second, we created both N- and C-terminal fusions (Figure 1A) in an attempt to mimic PCNA-K164 ubiquitination, as the PCNA N-terminus is situated on a separate but similar ridge to K164 and in a medial location between the faces, whereas the PCNA C-terminus and K164 reside on the same ridge, albeit on separate faces of the molecule (38). Hence, both fusions mimic distinct aspects of native K164-ubquitinated PCNA. Third, the C-terminal two Gly residues were removed from Ub in the fusion constructs to prevent protease cleavage (for Ub-PCNA) or further modifications (for PCNA-Ub). Fourth, no other tag is added to the fusion constructs to avoid unnecessary complications of the fusion proteins. Finally, an inducible Flp-FRT-mediated site-specific integration system was used in this study so that all stable transfectants are expected to be homogenous except for the genes of interest and so that target gene expression can be experimentally induced by adding Dox to the culture medium. Indeed, stable cell lines obtained from the same plasmid transfection displayed indistinguishable levels of fusion gene expression, which was under tight control of Dox (Figure 1B). It was noticed that the ectopic PCNA-K164R migrates with the same speed as endogenous PCNA (cf. lanes 5 and 6). Interestingly, PCNA-Ub appears to migrate slower and express at a slightly higher level than Ub-PCNA (cf. lanes 2 and 4). Nevertheless, the levels of ectopic fusion proteins are comparable with the level of endogenous PCNA, which facilitates this study.Figure 1.


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)

Establishment of stable cell lines expressing different forms of PCNA. (A) Schematic diagrams of PCNA•Ub fusion constructs. All ORFs contain a K164R mutation to prevent in vivo PCNA ubiquitination. In addition, the Ub-coding region is either fused to PCNA at the N-terminus (Ub-PCNA) or at the C-terminus (PCNA-Ub). All three ORFs are cloned into plasmid pcDNA5.0FRT/TO. (B) Ectopic expression of different forms of PCNA in stably transfected T-Rex-293 cells. Dox-induced expression of ectopic PCNA was analyzed by western blot using an anti-PCNA antibody, and the cellular β-actin serves as a loading control. PCNA-Ub and Ub-PCNA migrate differently, although they have the same molecular weight. (C) Polη and Rev1 preferentially bind to PCNA-Ub over PCNA. T-Rex-293/PCNA-Ub cells were transfected with GFP-Polη or GFP-mRev1 and incubated for 6 h followed by Dox treatment for 2 days. Immunoprecipitation was performed by GFP-Trap-A beads, and the products were analyzed by western blot. (D) The Ub-I44 residue is required for the enhanced affinity between PCNA-Ub and GFP-Polη. T-Rex-293/PCNA-Ub and T-Rex-293/PCNA-Ub-I44A cells were transfected with GFP-Polη, incubated for 6 h followed by Dox treatment for 2 days. Immunoprecipitation was performed by GFP-Trap-A beads, and the products were analyzed by western blot.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt542-F1: Establishment of stable cell lines expressing different forms of PCNA. (A) Schematic diagrams of PCNA•Ub fusion constructs. All ORFs contain a K164R mutation to prevent in vivo PCNA ubiquitination. In addition, the Ub-coding region is either fused to PCNA at the N-terminus (Ub-PCNA) or at the C-terminus (PCNA-Ub). All three ORFs are cloned into plasmid pcDNA5.0FRT/TO. (B) Ectopic expression of different forms of PCNA in stably transfected T-Rex-293 cells. Dox-induced expression of ectopic PCNA was analyzed by western blot using an anti-PCNA antibody, and the cellular β-actin serves as a loading control. PCNA-Ub and Ub-PCNA migrate differently, although they have the same molecular weight. (C) Polη and Rev1 preferentially bind to PCNA-Ub over PCNA. T-Rex-293/PCNA-Ub cells were transfected with GFP-Polη or GFP-mRev1 and incubated for 6 h followed by Dox treatment for 2 days. Immunoprecipitation was performed by GFP-Trap-A beads, and the products were analyzed by western blot. (D) The Ub-I44 residue is required for the enhanced affinity between PCNA-Ub and GFP-Polη. T-Rex-293/PCNA-Ub and T-Rex-293/PCNA-Ub-I44A cells were transfected with GFP-Polη, incubated for 6 h followed by Dox treatment for 2 days. Immunoprecipitation was performed by GFP-Trap-A beads, and the products were analyzed by western blot.
Mentions: Several considerations were taken into account when making the PCNA and Ub fusion (PCNA•Ub) constructs. First, the PCNA ORF used for fusions contains a K164R mutation to prevent in vivo PCNA ubiquitination at the K164 residue following UV irradiation. Second, we created both N- and C-terminal fusions (Figure 1A) in an attempt to mimic PCNA-K164 ubiquitination, as the PCNA N-terminus is situated on a separate but similar ridge to K164 and in a medial location between the faces, whereas the PCNA C-terminus and K164 reside on the same ridge, albeit on separate faces of the molecule (38). Hence, both fusions mimic distinct aspects of native K164-ubquitinated PCNA. Third, the C-terminal two Gly residues were removed from Ub in the fusion constructs to prevent protease cleavage (for Ub-PCNA) or further modifications (for PCNA-Ub). Fourth, no other tag is added to the fusion constructs to avoid unnecessary complications of the fusion proteins. Finally, an inducible Flp-FRT-mediated site-specific integration system was used in this study so that all stable transfectants are expected to be homogenous except for the genes of interest and so that target gene expression can be experimentally induced by adding Dox to the culture medium. Indeed, stable cell lines obtained from the same plasmid transfection displayed indistinguishable levels of fusion gene expression, which was under tight control of Dox (Figure 1B). It was noticed that the ectopic PCNA-K164R migrates with the same speed as endogenous PCNA (cf. lanes 5 and 6). Interestingly, PCNA-Ub appears to migrate slower and express at a slightly higher level than Ub-PCNA (cf. lanes 2 and 4). Nevertheless, the levels of ectopic fusion proteins are comparable with the level of endogenous PCNA, which facilitates this study.Figure 1.

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