Figure 2. Accumulation of SUMO2/3-conjugated proteins on chromatin during S phase depends on cyclin E.

(a) S-phase Xenopus egg extracts were supplemented or not with Ubc9dn, as in Fig. 1. The presence of SUMO-conjugated proteins was analysed by western blotting with anti-SUMO1 and anti-SUM02/3 antibodies, using replicating samples at the 60-min time-point. (b) Time-course of DNA replication in S-phase Xenopus egg extracts immunodepleted with anti-cyclin E (1) or control antibodies (2) or with Ctrl antibodies and supplemented with 5 μM SUMO1-VS (3), in the presence of α-[³³P]-dCTP. The graph represents the percentage of input DNA replicated at each indicated time-point. (c) Xenopus egg extracts described in Fig. 2b (2 μl) were immunoprobed with anti-SUMO2/3 and anti-cyclin E antibodies before addition of sperm nuclei and throughout the replication assays in cyclin E-depleted extract (1), control-depleted extract without (2) or with SUMO1-VS (3). (d) Aliquots of cytosol (Cyto), nuclei and chromatin fractions, corresponding to 2, 5 and 20 μl of extracts, respectively, were taken at the 45-min time-point after the addition of sperm nuclei from the replication reactions 1, 2 and 3, described in Fig. 2b and were immunoprobed with antibodies against SUMO2/3, cyclin E and Cdc6 (loading Ctrl).