Fig. 1

ATAD5-RLC is a robust PCNA unloader. a Deletion mutants of ATAD5 examined for PCNA unloading activity. Top diagram shows the locations of the reported motifs in human ATAD5. Lines between boxes represent predicted coiled-coil structures. Boundaries of motifs are denoted as amino-acid numbers. N-terminal deletion mutants of ATAD5 were fused to its putative NLS sequence located at its N-terminus (residues 1–32 of ATAD5). b The PCNA unloading activity is conferred by the C-terminal domain of ATAD5. The indicated ATAD5 deletion mutants were transiently expressed in ATAD5-depleted 293T cells. Chromatin-bound PCNA was analyzed by immunoblotting after chromatin fractionation. ATAD5 (ΔN692) exhibited PCNA unloading activity. c Quantification of chromatin-bound PCNA in (b). Values indicate relative chromatin-bound PCNA amount compared with the control chromatin (n = 3). Error bars indicate standard deviations in all quantification results. d Purification of ATAD5-RLC. Baculovirus encoding HIS-ATAD5 (ΔN692)-3XFLAG were co-infected to insect cells with a virus encoding RFC2–5. Pentameric ATAD5-RLC was purified from cell extracts through sequential application onto HIS, FLAG, and Heparin affinity columns. Coomassie-stained purified ATAD5-RLC has a stoichiometric amount of each subunit. e Schematic diagram of PCNA unloading assay. DNA substrates were prepared by annealing oligonucleotides to the 5′-biotinylated DNA. Substrate DNA was attached to the streptavidin-coated magnetic beads. PCNA was loaded to the substrate DNA by purified RFC. After washing, ATAD5-RLC was treated to the DNA-loaded PCNA. PCNA that remained on DNA was analyzed by immunoblotting. Error bars indicate standard deviations in all PCNA loading/unloading results. f ATAD5-RLC unloads PCNA. Indicated amounts of purified ATAD5-RLC, RFC, CTF18-RLC, and RAD17-RLC were treated to the DNA-loaded PCNA. 10-nucleotide-gap DNA (130-mer) was used for this assay. Unloading reaction contained 1 mM ATP. g Quantification of (f). Relative DNA-loaded PCNA amounts are indicated compared with the control reaction. Error bars indicate standard deviation (n = 3). See also Supplementary Fig. 1