Figure 4. Cyclin E (cyc E) rescue of the chromatin-associated pool of SUMO2/3 conjugates in cyc E-depleted Xenopus egg extracts depends on its SUMOylation.

(a) Wild-type (wt) [³⁵S]cyc E or [³⁵S]cyc E-KR, which were generated by in vitro transcription/translation in rabbit reticulocyte lysates, were incubated with a SUMOylation mix containing different recombinant SUMO proteins (His-SUMO2, SUMO1 or GST-SUMO1), as indicated. (b) Wt [³⁵S]cyc E or [³⁵S]cyc E-KR, which were obtained by messenger RNA translation in Cdk2-depleted Xenopus egg extracts, were purified by binding to GST-Cdk2. Kinase activity was checked by modification of the electrophoretic mobility of cyc E (top panel) and by histone H1 kinase assay (bottom panel). Radiolabelled cyc E and phosphorylated H1 were detected by phosphorimaging (c) Cyc E-immunodepleted S-phase Xenopus egg extracts (ΔE; Δc, mock depletion) were supplemented or not with wt [³⁵S]cyc E–Cdk2 (ΔE+Ewt) or [³⁵S]cyc E-KR–Cdk2 (ΔE+EKR) complexes and assayed for replication. Aliquots at the 45 min time-point after addition of sperm nuclei were used to measure the percentage of replicated DNA. (d) Replicating chromatin was prepared from 50 μl replication reactions (c) without SUMO1-VS and analysed by western blotting using antibodies against SUMO2/3, cyc E and Cdc6 (loading control). [³⁵S]cyc E was also detected by phosphorimaging. (e) Cdk2-immunodepleted S-phase Xenopus egg extracts were complemented with cyc E-[³⁵S]Cdk2 complexes that were translated and reconstituted in extracto, supplemented with either SUMO1-VS or with Ubc9dn and GST-SENP and assayed for replication. Aliquots at 30, 35 and 45 min after addition of sperm nuclei were used to measure the percentage of replicated DNA. (f) Replicating chromatin was prepared from 50 μl replication reactions (e) at the 25, 35 and 45 min time-points and analysed by western blotting using antibodies against SUMO2/3 and RPA32 (RPA). Ext: [³⁵S]Cdk2-complemented extract 0.1 μl. [³⁵S]Cdk2 was detected by phosphorimaging (left panel). Quantification of [³⁵S]Cdk2 on chromatin is shown in the middle panel and quantification of RPA in the right panel.