Journal: bioRxiv

Article Title: The Atad5 RFC-like complex is the major unloader of proliferating cell nuclear antigen in Xenopus egg extracts

doi: 10.1101/2023.08.28.555229

Figure Lengend Snippet: (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 experiments, including the one shown in ( C ).

Article Snippet: For immunodepletion of Rfc1, Atad5, or Asf1, 3 vol of an antiserum was bound to 1 vol of recombinant Protein A Sepharose Fast Flow (PAS; Cytiva, #17127902) at 4°C overnight.

Techniques: Plasmid Preparation, Incubation, Recombinant, Ligation, Serial Dilution, Western Blot, Agarose Gel Electrophoresis