Figure 5. ZRF1 interacts with XPC during UV-mediated DNA repair. (A) ZRF1 specifically binds to XPC. Control and FLAGZRF1-expressing cells were irradiated with UV light. After immunoprecipitation with FLAG-M2-agarose, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 4%. (B) Endogenous immunopreciptiations with ZRF1 antibodies. Precipitates were subjected to Western blotting, and blots were incubated with the indicated antibodies. Inputs correspond to 3%. (C) ZRF1 localization to DNA damage sites is dependent on XPC. Control fibroblasts and XPC patient fibroblasts expressing both mCherry-ZRF1 and DDB2-GFP were UV irradiated (100 J/m2) through a micropore membrane. Thirty minutes after irradiation, cells were preextracted and fixed. DNA damage sites were visualized by DDB2-GFP. (D) ZRF1 enriches at chromatin after UV irradiation in a XPC-dependent manner. Chromatin association assays with control fibroblasts (GM16248) and XPC patient fibroblasts (GM15983) 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 H2A-ubiquitin and ZRF1 abundance was calculated. Values are given as mean ± SEM (n = 3). (E) H2A ubiquitylation is not altered in XPA patient fibroblasts. Chromatin association assays with control fibroblasts (GM15876) and XPA fibroblasts (GM04312) after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. Relative intensities of H2A-ubiquitin/H2A, ZRF1 and RING1B abundance were measured. Values are given as mean ± SEM (n = 3). (F) Epistasis analysis of ZRF1 and XPC. The relative colony formation potential of control or ZRF1 knockdown cell lines treated with control (Control; ZRF1) or XPC siRNA (XPC; ZRF1+XPC) was analyzed at different UV doses. Gene knockdown was confirmed by Western blots (not depicted). Values are given as mean ± SEM (n = 3).
Figure 5.

ZRF1 interacts with XPC during UV-mediated DNA repair. (A) ZRF1 specifically binds to XPC. Control and FLAGZRF1-expressing cells were irradiated with UV light. After immunoprecipitation with FLAG-M2-agarose, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 4%. (B) Endogenous immunopreciptiations with ZRF1 antibodies. Precipitates were subjected to Western blotting, and blots were incubated with the indicated antibodies. Inputs correspond to 3%. (C) ZRF1 localization to DNA damage sites is dependent on XPC. Control fibroblasts and XPC patient fibroblasts expressing both mCherry-ZRF1 and DDB2-GFP were UV irradiated (100 J/m2) through a micropore membrane. Thirty minutes after irradiation, cells were preextracted and fixed. DNA damage sites were visualized by DDB2-GFP. (D) ZRF1 enriches at chromatin after UV irradiation in a XPC-dependent manner. Chromatin association assays with control fibroblasts (GM16248) and XPC patient fibroblasts (GM15983) 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 H2A-ubiquitin and ZRF1 abundance was calculated. Values are given as mean ± SEM (n = 3). (E) H2A ubiquitylation is not altered in XPA patient fibroblasts. Chromatin association assays with control fibroblasts (GM15876) and XPA fibroblasts (GM04312) after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. Relative intensities of H2A-ubiquitin/H2A, ZRF1 and RING1B abundance were measured. Values are given as mean ± SEM (n = 3). (F) Epistasis analysis of ZRF1 and XPC. The relative colony formation potential of control or ZRF1 knockdown cell lines treated with control (Control; ZRF1) or XPC siRNA (XPC; ZRF1+XPC) was analyzed at different UV doses. Gene knockdown was confirmed by Western blots (not depicted). Values are given as mean ± SEM (n = 3).

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