XB-IMG-118821
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Figure 8. DNA damage induces polyubiquitylation of PCNA. (A) UV-damaged sperm chromatin was isolated from X. laevis egg extract after 30, 60, 90, or 120 min. The associated proteins were analyzed by Western blotting using an antibody recognizing PCNA. The control portions of this experiment are reproduced from Fig. 1 A to facilitate comparison. (B) Duplicate samples from A contained 32P-dATP and DNA replication was measured as described in Materials and methods. Closed circles represent undamaged chromatin and open squares represent UV-damaged chromatin. (C) Undamaged chromatin, MMS-damaged chromatin, or UV-damaged chromatin was isolated from X. laevis egg extract after 45 min. Aphidicolin was added to 100 μg/ml at t = â10 min to the extract incubated with undamaged chromatin. The associated proteins were analyzed by Western blotting using an antibody recognizing PCNA. (D) X. laevis egg extract, preincubated with 100 μg/ml aphidicolin, was incubated with sperm chromatin and either buffer, 0.2 μg/μL T7-PCNA (rWT), or 0.2 μg/μL T7-PCNA (rK164R). Chromatin was isolated and analyzed by immunoblotting with an antibody recognizing either PCNA or the T7 tag. (E) X. laevis egg extract was preincubated with 100 μg/ml aphidicolin. Undamaged sperm chromatin was incubated in this extract which also contained buffer, 0.5 μg/μL His-tagged ubiquitin, or 0.5 μg/μL His-tagged ubiquitin (K63R). Chromatin was isolated and the bound proteins were analyzed by Western blotting with an antibody recognizing PCNA. Image published in: Leach CA and Michael WM (2005) Copyright © 2005, The Rockefeller University Press. Creative Commons Attribution-NonCommercial-ShareAlike license Larger Image Printer Friendly View |