Fig. 5.

Contributions of the canonical PIP motif to PCNA binding. (A) Generation of combined fusions. GST was fused to a peptide spanning amino acids 320 to 388 of human ESCO2 containing both box C and the downstream canonical PIP-like motif at amino acids 374 to 377. A derivative in which the PIP-like motif was mutated (ESCO2 Q374A, I377A) was also purified. (B) Surface plasmon resonance analysis. The GST-fusion peptides in A were used to test PCNA binding using SPR, in which GST fusions were bound by anti-GST antibody and recombinant trimeric PCNA was used as an analyte. Shown are the background-subtracted sensorgrams (Left) and binding curves (Right) for p21 (positive control), GST (negative control), and the indicated fusions. Binding constants are shown at Right. The χ² residuals are represented as RU², an indication of curve fitting. Vertical blue lines indicate K_D. (C) Contribution of the Eco1 PIP box to PCNA interaction in S. cerevisiae. GST was fused to the first 33 amino acids of S. cerevisiae Eco1p (GST-Eco1-PIP) for use in a pull-down assay (as in Fig. 4). Disruption of the PIP motif by 2 amino acid substitutions (Q18A, L21A = GST-Eco1-PIP-AA) disrupted the ability to pull down PCNA from whole cell yeast extract, as indicated by immunoblot analysis. A parallel gel was Coomassie stained, showing the bead-associated GST fusions. WCE, whole cell yeast extract.