Fig. 5.

ORC3 and ORC5 multi-mono-ubiquitylation is essential for origin firing. a U2OS cells were co-transfected with His₆-tagged wild type (WT) ORC3 and ORC5 or lysine-less versions (0 K, see Supplementary Fig. 10a) and HA-ubiquitin, as indicated. Two days post-transfection, cells were lysed in denaturing conditions for His₆-tagged protein purification. Expression and ubiquitin conjugation of the isolated proteins were monitored by western blotting. b U2OS cells were co-transfected with Myc-tagged (WT or 0 K) ORC3 and ORC5 or EGFP and Flag-ORC2. Two days after transfection, cell lysates were Flag-immunoprecipitated to purify the ORC subunit, and the association of co-expressed proteins was analysed by western blotting. Expression of Myc-tagged proteins in input extracts was also monitored. c U2OS cells were transfected with ORC3 and ORC5 (WT or 0 K), Myc-geminin or empty vector and with a puromycin-resistance plasmid. The next day, transfected cells were selected in puromycin medium for 2 days, and grown for another day without selection. Cells were then pulsed with BrdU for 15 min and analysed by flow cytometry (upper panels). BrdU incorporation fluorescence signal was quantified from three independent experiments (lower left panel). Expression of ectopic proteins and endogenous PCNA was assessed by western blotting (lower right panels). ns, non-significant; *p < 0.01. d U2OS cells were treated as in c. Chromatin and soluble fractions were isolated and analysed by western blotting with antibodies against the indicated proteins. e DNA fibre stretching analysis. U2OS cells were treated as in c. Cells were incubated with IdU (15 min; red) followed by CldU (15 min; green) and processed for DNA spreading. Representative images are shown. Scale bars, 5 μm. The length of CldU tracks after the IdU signal was measured. More than 200 measurements are shown from two independent experiments. Red bars indicate median values. ns, non-significant; *p < 0.01