Figure 3.

α-Amanitin induction of Pol II ubiquitination is DNA-dependent. Ubiquitination reactions with nuclear extracts from aphidicolin-treated cells and His-Ub were titrated against increasing amounts of pUC118-296 (0–2.4 μg, lanes 3–12), and the effect of α-amanitin (α-am) was assayed. Reactions were analyzed as described in the legend of Fig. 1, and immunoblots of total (Upper) and Ni-NTA-bound proteins (Lower) against N20 are shown. Lanes 1, untreated extracts; lanes 2, ubiquitination reaction containing 1 μg of DNA, incubated in the absence of both His-Ub and α-amanitin.