FIGURE 4.

Rad5 catalyzes the extension of a Ub signal on PCNA. A, diagram comparing the isopeptide linkage of monoUb-PCNA (top panel) and the chemically synthesized analog, monoUb*-PCNA (bottom panel) (adopted from Ref. 30). B, FLAG-PCNA and H₆-Ub(G76C) (lane 1) were coupled using dichloroacetone (lane 2) to produce monoUb*-PCNA. Unreacted H₆-Ub and cross-linked H₆-Ub dimers were removed by gel filtration (lane 3). C, monoUb_n*-PCNA reaction products (n = 0–3) were separated using native gel electrophoresis (lane 1) and visualized with Coomassie Blue. The mixture of products was separated by nickel-nitrilotriacetic acid chromatography to yield monoUb₃*-PCNA (lane 2) and monoUb_1–2*-PCNA (lane 3). D, PCNA (lanes 1–6) or monoUb₃*-PCNA (lanes 7–15) were incubated in conjugation reactions containing E1, Ubc13-Mms2, and Ub(K63R), with or without Rad5, as indicated. The products were separated by SDS-PAGE and visualized with Coomassie Blue. E, reactions were as in D, except that monoUb₃*-PCNA was first loaded onto DNA by RFC (lanes 3 and 4), and the PCNA species were detected by immunoblotting with anti-FLAG antibodies.