Fig. 2

Active ATAD5-RLC formation requires functional RFC2–5 binding motif. a Constructs to identify the RFC2–5 binding motif (RBM). The positions of the deletions and point mutations (CM1–4) are indicated. Four conserved five-amino-acid stretches in RBM were mutated and denoted as CM1–4 (see Supplementary Fig. 2b). b–d ATAD5 (1602–1844) is a RBM. The indicated ATAD5 fragments were transiently expressed in 293T cells and immunoprecipitated using anti-FLAG beads. Bindings of RFC2–5 were monitored by co-immunoprecipitation of RFC4 or RFC5. b ATAD5 (1602–1719) is important for RFC2–5 binding. ATAD5 (1602–1844) bound to RFC2–5, but ATAD5 (1720–1844) did not. c ATAD5 (1720–1844) is also necessary for RFC2–5 binding. d E¹⁷¹³ALSF¹⁷¹⁷ of ATAD5 is important for RFC2–5 binding. The CM1 mutant, E¹⁷¹³ALSF¹⁷¹⁷ to AAGGG, was severely defective in RFC2–5 binding. e–g Proper RFC2–5 binding to ATAD5 is crucial for PCNA unloading. PCNA unloading activity of the indicated ATAD5 variants were examined as described in Fig. 1b. e RBM-deleted ATAD5 (693–1719) is defective in PCNA unloading. Graph shows relative amount of PCNA on the chromatin (n = 3). f ATAD5 (CM1), which is defective in RFC2–5 binding, is severely defective in PCNA unloading. Graph shows relative amount of PCNA on the chromatin (n = 3). g RBM-deleted ATAD5 (693–1719) fails to unload PCNA in vitro. In total, 1.4 kbps DNA was used for this assay. Graph shows relative PCNA amounts remained on DNA after unloading reaction (n = 2). See also Supplementary Fig. 2