FIGURE 2.

The B+4 residue can be partially compensated for by residues upstream of the PIP box in Xic1 and Set8. In A: Top, sequence comparison of Xic1 PIP degron with upstream basic residues and the various mutants examined. Bottom, graph showing the percentage of in vitro translated ³⁵S-labeled Xic1 remaining after it was added to HSS in the presence of 5 ng/μl MMS-damaged plasmid. Reactions were stopped at the indicated time points, and the amount of Xic1 remaining was quantified by autoradiography. Results from three independent experiments were averaged and graphed. Bars represent the standard error of the mean. In B: Top, sequence alignment of Set8 PIP degron with upstream residues and the various mutants examined. Bottom, HSS was supplemented with 5 ng/μl MMS plasmid and 50 nm human Set8^WT, Set8^ΔPIP, or Set8^B+3/4A. At the indicated times, samples were blotted for endogenous Cdt1 or Set8. C, HSS was supplemented with immobilized 1-kb MMS DNA and 2 mg/ml methyl ubiquitin. Buffer and 50 nm Set8^WT, Set8^ΔPIP, or Set8^B+3/4A was also added, and after 10 min, chromatin was recovered from the extract and washed, and the indicated proteins were visualized by Western blotting.