Figure 5. Cdt1 and CRL4^Cdt2 independently associate with PCNA loaded on DNA through PIP box in vitro.

(A) Purified CRL4^Cdt2(WT), CRL4^Cdt2(PIP-3A), and Cdt1 proteins. (B) Cdt1, but not CRL4^Cdt2, associates with free PCNA. The purified Cdt1 and CRL4^Cdt2 proteins were fixed on anti-FLAG–resins and incubated with PCNA for 2h at 4°C and the bound PCNA was analysed. The relative amounts of bound PCNA were shown, normalized with FLAG signal, and the level on Cdt1-3FLAG beads set to 1.0. Error bars represent SD from three independent experiments. (C) The ncDNA beads (DNA+) or control beads (DNA−) were loaded with PCNA by RFC or not. Then, beads were incubated with the purified Cdt1 or CRL4^Cdt2, and the bound proteins were recovered for Western blotting. The amounts of PCNA (as a trimer) loaded on plasmid DNA and the amounts of bound Cdt1-3FLAG and Cdt2-3FLAG were measured and shown (f mol). The number of bound Cdt1 and Cdt2 on PCNA trimer was shown (Cdt1/PCNA and Cdt2/PCNA). Error bars represent SD from independent experiments (n = 3 or n > 3). (D) CRL4^Cdt2(WT) and CRL4^Cdt2(PIP-3A) were incubated with control beads, ncDNA beads, or ncDNA beads which had been pre-incubated with RFC alone or together with PCNA for 1 h at 4°C. Beads were recovered and bound proteins were analysed.