Figure 4.

Isopeptidase activity of USP7 mutants for monoubiquitinated PCNA and ubiquitin chains. A–C, isopeptidase activity for monoubiquitinated PCNA. The reactions were performed as described in Fig. 1E. The amounts of unmodified PCNA generated in the reactions with the indicated concentrations of ^hisΔN (A), ^hisΔN/PL2 (B), or ^hisΔN/UL2 (C) are shown. D–F, isopeptidase activity for Lys⁶³-linked diubiquitin. The reactions were performed as described in Fig. 2A. The amounts of the remaining Lys⁶³-linked diubiquitin in the reactions with the indicated concentrations of ^hisΔN (D), ^hisΔN/PL2 (E), or ^hisΔN/UL2 (F) are shown. The averages of two independent experiments were plotted. In most cases, the error bars were smaller than the symbols. G, preferential cleavage of the PCNA-ubiquitin linkage of monoubiquitinated or polyubiquitinated PCNA over the Lys⁶³-diubiquitin linkage. The catalytic rate for the PCNA-ubiquitin linkage was divided by that for Lys⁶³-linked diubiquitin. H, Lys⁶³-linked diubiquitin (top panels), tetra-ubiquitin (middle panels), or hexa-ubiquitin (bottom panels) (240 fmol) as Lys⁶³ linkage was incubated with the indicated concentrations of ^hisUSP7 or ^hisΔN for 10 min. The reaction products were analyzed by Western blotting with an anti-ubiquitin mAb. I, a mixture of Lys⁶³-linked polyubiquitin chains was incubated with the indicated concentration of ^hisUSP7 or ^hisΔN for the indicated times. The reaction products were analyzed by Western blotting with an anti-ubiquitin mAb.