Fig. 4.

OBI1 catalyses ORC3 and ORC5 multi-mono-ubiquitylation. a U2OS cells were co-transfected with the indicated Myc-Flag (MF)-tagged ORC subunits and HA-tagged ubiquitin. When indicated ( + ), Myc-tagged OBI1 was also co-expressed. Two days post-transfection, cells were lysed in NEM-containing lysis buffer and tagged proteins were purified by anti-Flag immunoprecipitation. Expression and ubiquitin conjugation of the immunoprecipitates were monitored by western blotting, as indicated. Myc-OBI1 expression was also analysed in input extracts. b, c U2OS cells were co-transfected with His₆-Flag-ORC3 (HF-ORC3) or His₆-Flag-ORC5 (HF-ORC5) and either wild type (WT) or inactive (CS) Myc-OBI1 along with HA-ubiquitin as indicated. ORC3/5 ubiquitylation was detected by purification in denaturing conditions on nickel beads (Ni-NTA). Expression and ubiquitin conjugation of the isolated proteins were monitored by western blotting. Myc-OBI1 expression was also analysed in input extracts. d, e U2OS cells were transfected with Mock or OBI1-specific siRNAs. The next day, cells were co-transfected with Myc-Flag (MF)-tagged ORC3 or ORC5 and HA-tagged ubiquitin, as indicated. Ubiquitylation was detected 2 days later as in a. Expression of endogenous OBI1 was monitored in input extracts by western blotting. f, g OBI1 in vitro assay. U2OS cells were transfected with wild type Flag-OBI1 (WT) or inactive mutant (CS) for two days. Immunoprecipitated OBI1 (IP: Flag) was incubated with in vitro translated Myc-ORC3 or -ORC5 along with E1, E2 (UbcH5a), ubiquitin and ATP for 30 min. at 37 °C. Tagged proteins were detected by western blotting as indicated. h OBI1 in vitro assay performed as in f, g, but with wild type (WT) or lysine-less (0 K) ubiquitin. Tagged proteins were detected by western blotting as indicated