Figure 7. ZRF1 facilitates the assembly of the UV–DDB–CUL4A E3 ligase complex. (A) ZRF1 displaces RING1B from chromatin during NER. Chromatin association assays of control and ZRF1 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 H2A ubiquitin and RING1B abundance was calculated. Values are given as mean ± SEM (n = 3). (B) ZRF1 regulates chromatin association of CUL4A and CUL4B. Chromatin association assays of control and ZRF1 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 CUL4B and CUL4A abundance was calculated. Values are given as mean ± SEM (n = 3). (C) ZRF1 regulates CUL4A association with H2AX containing nucleosomes. Control cells and ZRF1 knockdown cells expressing FLAGH2AX were irradiated with UV. 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 3%. (D) Knockdown of ZRF1 modulates CUL4A association with DDB2. Control cells and ZRF1 knockdown cells expressing FLAGDDB2 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 3%. (E) Assembly of the UV–DDB–CUL4A E3 ligase is facilitated by ZRF1. Control cells and ZRF1 knockdown HEK293T cells expressing HARBX1 were irradiated with UV light. After immunoprecipitation with HA-specific antibodies the precipitated material was subjected to Western blotting, and blots were incubated with the indicated antibodies. Inputs correspond to 5%. (F) ZRF1 competes with CUL4B and RING1B for DDB2 binding in vitro. GFP and GFP-DDB2 immobilized on beads were incubated with equimolar amounts of purified DDB1, CUL4B, and RING1B and increasing amounts of ZRF1. ZRF1 levels were doubled stepwise reaching an eightfold molar excess of ZRF1 over the other components (relative molarity ZRF1: DDB1–CUL4B–RING1B; lane 3, 1:1; lane 4, 2:1; lane 5, 4:1; lane 6, 8:1). Precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 10%. (G) ZRF1 does not compete with CUL4A and RBX1 for binding to DDB1–DDB2. GFP and GFP-DDB2 immobilized on beads were incubated with equimolar amounts of purified DDB1, CUL4A and RBX1 and increasing amounts of ZRF1. ZRF1 levels were doubled stepwise reaching an eightfold molar excess of ZRF1 (relative molarity ZRF1: DDB1–CUL4A–RBX1; lane 3, 1:1; lane 4, 2:1; lane 5, 4:1; lane 6, 8:1). Precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 10%. (H) ZRF1 mediates the formation of the UV-DDB-CUL4A complex in vitro. GFP and GFP-DDB2 were coupled to beads and incubated with CUL4B, DDB1 and RING1B. After washing, GFP and GFP-DDB2 (UV–RING1B complex) beads were incubated with an estimated fivefold excess of purified CUL4A and RBX1 (lanes 1–3) over the retained UV–RING1B complex. Simultaneously, ZRF1 (lanes 1 and 3) or GST (lane 2) was added to the incubations in equimolar amounts. The precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 5%.
Figure 7.

ZRF1 facilitates the assembly of the UV–DDB–CUL4A E3 ligase complex. (A) ZRF1 displaces RING1B from chromatin during NER. Chromatin association assays of control and ZRF1 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 H2A ubiquitin and RING1B abundance was calculated. Values are given as mean ± SEM (n = 3). (B) ZRF1 regulates chromatin association of CUL4A and CUL4B. Chromatin association assays of control and ZRF1 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 CUL4B and CUL4A abundance was calculated. Values are given as mean ± SEM (n = 3). (C) ZRF1 regulates CUL4A association with H2AX containing nucleosomes. Control cells and ZRF1 knockdown cells expressing FLAGH2AX were irradiated with UV. 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 3%. (D) Knockdown of ZRF1 modulates CUL4A association with DDB2. Control cells and ZRF1 knockdown cells expressing FLAGDDB2 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 3%. (E) Assembly of the UV–DDB–CUL4A E3 ligase is facilitated by ZRF1. Control cells and ZRF1 knockdown HEK293T cells expressing HARBX1 were irradiated with UV light. After immunoprecipitation with HA-specific antibodies the precipitated material was subjected to Western blotting, and blots were incubated with the indicated antibodies. Inputs correspond to 5%. (F) ZRF1 competes with CUL4B and RING1B for DDB2 binding in vitro. GFP and GFP-DDB2 immobilized on beads were incubated with equimolar amounts of purified DDB1, CUL4B, and RING1B and increasing amounts of ZRF1. ZRF1 levels were doubled stepwise reaching an eightfold molar excess of ZRF1 over the other components (relative molarity ZRF1: DDB1–CUL4B–RING1B; lane 3, 1:1; lane 4, 2:1; lane 5, 4:1; lane 6, 8:1). Precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 10%. (G) ZRF1 does not compete with CUL4A and RBX1 for binding to DDB1–DDB2. GFP and GFP-DDB2 immobilized on beads were incubated with equimolar amounts of purified DDB1, CUL4A and RBX1 and increasing amounts of ZRF1. ZRF1 levels were doubled stepwise reaching an eightfold molar excess of ZRF1 (relative molarity ZRF1: DDB1–CUL4A–RBX1; lane 3, 1:1; lane 4, 2:1; lane 5, 4:1; lane 6, 8:1). Precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 10%. (H) ZRF1 mediates the formation of the UV-DDB-CUL4A complex in vitro. GFP and GFP-DDB2 were coupled to beads and incubated with CUL4B, DDB1 and RING1B. After washing, GFP and GFP-DDB2 (UV–RING1B complex) beads were incubated with an estimated fivefold excess of purified CUL4A and RBX1 (lanes 1–3) over the retained UV–RING1B complex. Simultaneously, ZRF1 (lanes 1 and 3) or GST (lane 2) was added to the incubations in equimolar amounts. The precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 5%.

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