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Proliferating cell nuclear antigen (PCNA) is a homo-trimeric clamp complex that serves as the molecular hub for various DNA transactions, including DNA synthesis and post-replicative mismatch repair. Its timely loading and unloading are critical for genome stability. PCNA loading is catalyzed by Replication factor C (RFC) and the Ctf18 RFC-like complex (Ctf18-RLC), and its unloading is catalyzed by Atad5/Elg1-RLC. However, RFC, Ctf18-RLC, and even some subcomplexes of their shared subunits are capable of unloading PCNA in vitro, leaving an ambiguity in the division of labor in eukaryotic clamp dynamics. By using a system that specifically detects PCNA unloading, we show here that Atad5-RLC, which accounts for only approximately 3% of RFC/RLCs, nevertheless provides the major PCNA unloading activity in Xenopus egg extracts. RFC and Ctf18-RLC each account for approximately 40% of RFC/RLCs, while immunodepletion of neither Rfc1 nor Ctf18 detectably affects the rate of PCNA unloading in our system. PCNA unloading is dependent on the ATP-binding motif of Atad5, independent of nicks on DNA and chromatin assembly, and inhibited effectively by PCNA-interacting peptides. These results support a model in which Atad5-RLC preferentially unloads DNA-bound PCNA molecules that are free from their interactors.
Figure 1. Quantification of the concentration of RFC and RLCs in NPE.A and B, immunoprecipitation was performed from NPE using preimmune- (“mock”, lane 1), Rfc3- (lane 2), Rfc1- (lane 3), Ctf18- (lane 4), Atad5- (lane 5), or Rad17-antibodies (lane 6), and 0.25 μl each of the supernatant (A) and the IP fractions corresponding to 2.5 μl each of NPE (B) were separated by SDS-PAGE alongside a serial dilution series of the supernatant of mock-IP NPE (A) or the indicated amounts of recombinant His6-Rfc3 (B), followed by immunoblotting with indicated antibodies. Orc2 serves as a loading control. (∗) Cross-reacting band. C, the estimated concentrations of RFC and RLCs in NPE were plotted on a graph. Filled circles represent individual data from two independent experiments, including the one shown in (B).
Figure 2. Atad5-RLC is the major PCNA unloader in Xenopus egg extracts.A, a schematic diagram of the PCNA unloading assay. Singly biotinylated plasmid carrying a nick was bound to Sepharose beads and incubated with recombinant human PCNA and RFC to load PCNA on DNA. After nick ligation and extensive wash, the resulting PCNA-DNA complex was transferred into Xenopus egg extracts to examine PCNA unloading. B, 0.25 μl each of mock-treated (lane 1), Rfc3-depleted (lane 2), Rfc1-depleted (lane 3), Ctf18-depleted (lane 4), Atad5-depleted (lane 5), Rad17-depleted NPE (lane 6), and a serial dilution series of mock-treated NPE (lanes 7–11) were analyzed by immunoblotting with indicated antibodies. Orc2 serves as a loading control. C, PCNA loaded onto immobilized DNA was incubated in NPE described in (B). Untreated DNA separated by agarose gel electrophoresis in the presence of ethidium bromide, a quantitative immunoblot of hPCNA and Rfc3 in the bead-bound fractions, and linearized DNA (treated with XmnI) separated by agarose gel electrophoresis are presented along with the percentage of covalently-closed plasmids, the estimated numbers of DNA-bound PCNA molecules per plasmid, and the amount of bead-bound DNA. D, the average numbers of DNA-bound PCNA per plasmid at the 10-min time points were plotted into a graph. Filled circles represent individual data from two independent experiments, including the one shown in (C), except for the ‘Input’ and ‘mock’ samples, which were triplicated.
Figure 3. The Walker A motif of ATAD5 is required for PCNA unloading. A, a schematic diagram of the human ATAD5 expression construct is presented. Monomeric Azami-Green (mAG) and a FLAG epitope were fused at the N- and C-termini, respectively. The Walker A mutant of ATAD5 was made by substituting the conserved lysine 1138 residue with glutamic acid. B, 250 ng each of purified wild-type (WT) and the Walker A mutant (K1138E) ATAD5-RLCs were separated by SDS-PAGE and stained with Coomassie brilliant blue R-250. (∗) indicates unrelated proteins co-purified with ATAD5-RLC. C, the PCNA unloading assay in mock-treated HSS supplemented with buffer (lanes 2 and 3), Atad5-depleted HSS supplemented with buffer (lanes 4 and 5) or 12 nM ATAD5-RLC (lanes 6 and 7), or Rfc3-depleted HSS supplemented with buffer (lanes 8 and 9) or 12 nM ATAD5-RLC (lanes 10 and 11). An immunoblot of DNA-bound PCNA (top) and linearized DNA separated by agarose gel electrophoresis (bottom) are presented, along with the estimated numbers of PCNA molecules per DNA and the amounts of DNA. D, the average numbers of DNA-bound PCNA per plasmid at the 15-min time points were plotted into a graph. Filled circles represent individual data from two independent experiments, including the one shown in (C). E, the PCNA unloading assay in mock-treated HSS supplemented with buffer (lanes 2 and 3), or Atad5-depleted HSS supplemented with buffer (lanes 4 and 5), 12 nM ATAD5WT-RLC (lanes 6 and 7), or ATAD5K1138E-RLC (lanes 8 and 9). An immunoblot of DNA-bound PCNA (top) and linearized DNA (bottom) are presented, along with the estimated numbers of PCNA molecules per DNA and the amounts of DNA. F, the average numbers of DNA-bound PCNA per plasmid at the 15-minute time points were plotted into a graph. Filled circles represent individual data from three independent experiments, including the one shown in (E).
Figure 4. Single-strand nicks and chromatin assembly do not detectably affect PCNA unloading in Xenopus egg extracts. A, the PCNA unloading assay in mock-treated HSS supplemented with buffer (lanes 3–5) or Nb.BtsI (lanes 6–8), or Atad5-depleted HSS supplemented with buffer (lanes 9–11) or Nb.BtsI (lanes 12–14), presented with the input samples before (lane 1) and after nick ligation (lane 2). Untreated DNA separated by agarose gel electrophoresis in the presence of ethidium bromide (top), a quantitative immunoblot of DNA-bound PCNA (middle), and linearized DNA separated by agarose gel electrophoresis (bottom) are shown, along with the estimated numbers of PCNA molecules per DNA and the amounts of DNA. B, the average numbers of DNA-bound PCNA per plasmid at the 10-min time points were plotted into a graph. Filled circles represent individual data from two independent experiments, including the one shown in (A). C, 2 μl each of mock-treated (lane 1) and Asf1-depleted HSS (lane 2) were analyzed by immunoblotting with indicated antibodies, along with a serial dilution series of mock-treated HSS (lanes 3–8). Orc2 serves as a loading control. D, the PCNA unloading assay in HSS described in (C). Untreated DNA (top), a quantitative immunoblot of DNA-bound PCNA (middle), and linearized DNA (bottom) are presented, along with the estimated numbers of DNA-bound PCNA molecules per plasmid and the amounts of bead-bound DNA. E, the average numbers of DNA-bound PCNA per plasmid at the 10-min time points were plotted into a graph. Filled circles represent individual data from two independent experiments, including the one shown in (D).
Figure 5. PCNA-binding peptides inhibit PCNA unloading. A, the PCNA unloading assay in HSS supplemented with the solvent (H2O, lanes 2 and 3), wild-type (p21WT, lanes 4 and 5), PIP-mutant (p21pip, lanes 6 and 7), or sequence-scrambled p21 peptides (p21jumbled, lanes 8 and 9). A quantitative immunoblot of DNA-bound PCNA (top) and linearized DNA separated by agarose gel electrophoresis (bottom) are presented, along with the estimated numbers of PCNA molecules per DNA and the amounts of DNA. B, the average numbers of DNA-bound PCNA per plasmid at the 15-min time points were plotted into a graph. Filled circles represent individual data from two independent experiments, including the one shown in (A). C, the PCNA unloading assay in HSS supplemented with the solvent (H2O, lanes 2 and 3), wild-type p21 peptide (p21WT, lanes 4 and 5), wild-type (Msh6WT, lanes 6 and 7), PIP-mutant (Msh6pip, lanes 8 and 9), or sequence-scrambled Msh6 peptides (Msh6jumbled, lanes 10 and 11). A quantitative immunoblot of DNA-bound PCNA (top) and linearized DNA (bottom) are presented, along with the estimated numbers of PCNA molecules per DNA and the amounts of DNA. D, the average numbers of DNA-bound PCNA per plasmid at the 15-min time points were plotted into a graph. Filled circles represent individual data from two independent experiments, including the one shown in (C).
