Figure 4.
Figure 4. Function of ZRF1 in UV-mediated DNA repair. (A) ZRF1 is tethered to chromatin in a RING1B-dependent manner. Chromatin association assays of control and RING1B knockdown HEK293T cell lines after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative ZRF1 abundance was calculated. Values are given as mean ± SEM (n = 3). (B) The ubiquitin-binding domain (UBD) is important for tethering ZRF1 to chromatin after UV irradiation. HEK293T cells expressing FLAGZRF1 and FLAGZRF1-ΔUBD were irradiated with UV light, and chromatin was isolated at the indicated time points. De-cross-linked material was subjected to Western blotting and blots were incubated with FLAG-antibody. The relative FLAGZRF1 abundance was calculated. Values are given as mean ± SEM (n = 4). (C and D) ZRF1 localizes to DNA damage sites after UV irradiation. MRC5 fibroblasts expressing mCherry-ZRF1 were UV irradiated (100 J/m2) through a micropore membrane (+ UV) 24 h after transfection. 30 min after irradiation, cells were preextracted and fixed. DNA damage sites were visualized by staining with XPC (C) or XPA (D) antibody. The colocalization of ZRF1 with XPC amounts to 88% ± 1%. The colocalization of ZRF1 with XPA amounts to 73% ± −3%. Nonirradiated control and quantification of the ZRF1 localization at the damage sites are represented in Fig. S4 A. Bar, 10 µm. (E) Inhibition of RING1B affects recruitment of ZRF1 to DNA damage sites. MRC5 fibroblasts expressing mCherry-ZRF1 were treated with PRT4165 or DMSO. Cells were UV-irradiated (100 J/m2) through a micropore membrane. 30 min after irradiation cells were preextracted and fixed. DNA damage sites were visualized by XPC antibody staining. ZRF1 localization to DNA lesions after treatment with DMSO or PRT4165 was quantified (Fig. S4 B). Bar, 10 µm. (F) Depletion of CUL4B impacts H2A ubiquitylation and ZRF1 recruitment. Chromatin association assays of UV irradiated HEK293T cells treated with siRNAs (control, CUL4B). De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative H2A-ubiquitin and ZRF1 abundance was calculated. Values are given as mean ± SEM (n = 3). (G) Tethering of ZRF1 to chromatin depends on DDB2 during NER. Chromatin association assays in control fibroblasts (GM15876) and XPE (DDB2) fibroblasts (GM01389) after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative RING1B and ZRF1 abundance was calculated. Values are given as mean ± SEM (n = 3).

Function of ZRF1 in UV-mediated DNA repair. (A) ZRF1 is tethered to chromatin in a RING1B-dependent manner. Chromatin association assays of control and RING1B knockdown HEK293T cell lines after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative ZRF1 abundance was calculated. Values are given as mean ± SEM (n = 3). (B) The ubiquitin-binding domain (UBD) is important for tethering ZRF1 to chromatin after UV irradiation. HEK293T cells expressing FLAGZRF1 and FLAGZRF1-ΔUBD were irradiated with UV light, and chromatin was isolated at the indicated time points. De-cross-linked material was subjected to Western blotting and blots were incubated with FLAG-antibody. The relative FLAGZRF1 abundance was calculated. Values are given as mean ± SEM (n = 4). (C and D) ZRF1 localizes to DNA damage sites after UV irradiation. MRC5 fibroblasts expressing mCherry-ZRF1 were UV irradiated (100 J/m2) through a micropore membrane (+ UV) 24 h after transfection. 30 min after irradiation, cells were preextracted and fixed. DNA damage sites were visualized by staining with XPC (C) or XPA (D) antibody. The colocalization of ZRF1 with XPC amounts to 88% ± 1%. The colocalization of ZRF1 with XPA amounts to 73% ± −3%. Nonirradiated control and quantification of the ZRF1 localization at the damage sites are represented in Fig. S4 A. Bar, 10 µm. (E) Inhibition of RING1B affects recruitment of ZRF1 to DNA damage sites. MRC5 fibroblasts expressing mCherry-ZRF1 were treated with PRT4165 or DMSO. Cells were UV-irradiated (100 J/m2) through a micropore membrane. 30 min after irradiation cells were preextracted and fixed. DNA damage sites were visualized by XPC antibody staining. ZRF1 localization to DNA lesions after treatment with DMSO or PRT4165 was quantified (Fig. S4 B). Bar, 10 µm. (F) Depletion of CUL4B impacts H2A ubiquitylation and ZRF1 recruitment. Chromatin association assays of UV irradiated HEK293T cells treated with siRNAs (control, CUL4B). De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative H2A-ubiquitin and ZRF1 abundance was calculated. Values are given as mean ± SEM (n = 3). (G) Tethering of ZRF1 to chromatin depends on DDB2 during NER. Chromatin association assays in control fibroblasts (GM15876) and XPE (DDB2) fibroblasts (GM01389) after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative RING1B and ZRF1 abundance was calculated. Values are given as mean ± SEM (n = 3).

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