Azuma Y , Arnaoutov A , Dasso M .

	Figure 1. Chromatin-dependent SUMO conjugation of mitotic substrates. CSF extracts were supplemented with histidine-tagged SUMO-1 (final concentration 30 ng/μl) and with the indicated number of sperm nuclei. After formation of condensed metaphase chromosomes (90 min after the start of the reaction), CSF arrest was released into interphase by addition of 0.6 mM CaCl2. The SUMO-1–conjugated protein profile throughout the reaction was analyzed by Western blotting with affinity-purified anti-SUMO-1 antibodies. There were a series of strong M-phase–specific SUMO-1 conjugates observed in the reaction containing 10,000 sperm nuclei/μl (bottom, indicated by an asterisk). These bands could also be weakly observed in the reaction containing 1,000 sperm nuclei/μl, but were absent in the reaction without nuclei. These bands were lost after the extracts entered interphase. These characteristics indicated that the bands resulted from modification of mitotic substrates in a chromatin-dependent manner.
	Figure 2. DNA topoisomerase II is modified by SUMO-2/3 in M-phase extracts. (A) CSF extracts containing 10,000 sperm nuclei/μl were incubated at 23°C for 60 min with or without exogenous 30 ng/μl His-SUMO-1 or 150 ng/μl dnUbc9. Aliquots were analyzed by immunoblotting with anti–Xenopus topoisomerase II, anti-SUMO-1, and anti-SUMO-2/3 antibodies. (B) CSF extract containing 10,000 sperm nuclei/μl was incubated 60 min at 23°C. The reaction was split, and half was driven into interphase though the addition of 0.6 mM CaCl2. Each reaction was incubated at 23°C for an additional 30 min. Chromatin was isolated from both mitotic and interphase extracts, as described in Materials and methods. The chromatin was subjected to Western blotting with the following antibodies: (1) monoclonal anti–human topoisomerase IIα/β (DNATop2AB); (2) monoclonal anti–human topoisomerase IIα; (3) polyclonal anti– human topoisomerase IIα; (4) anti–Xenopus topoisomerase II; and (5) anti-SUMO-2/3, respectively. Note that topoisomerase IIα is essentially the sole form of topoisomerase II in egg extracts. (C) CSF extracts containing the indicated number of sperm nuclei/μl were incubated at 23°C for 60 min. Equal amounts of isolated chromatin were subjected to SDS-PAGE and analyzed by Western blotting using anti-SUMO-2/3 and anti-topoisomerase II antibodies. Asterisks indicate SUMO-conjugated forms of topoisomerase II in all panels.
	Figure 3. Topoisomerase II is modified by SUMO-2/3 during mitosis in unperturbed cycling extracts. Xenopus cycling egg extracts were supplemented with 2,000 sperm nuclei/μl and incubated at 23°C. Samples were taken from extracts at the indicated times. Aliquots of each sample were directly subjected to SDS-PAGE and Western blotting analysis to determine cyclin B abundance (fourth panel) and phosphorylated ERK1 levels (bottom). Chromatin was separately prepared from samples taken at each time point, as described in Materials and methods. The chromatin preparations were analyzed by Western blotting with antibodies against topoisomerase II (top), SUMO-2/3 (second panel), and SUMO-1 (middle). Aliquots were also removed at the indicated times and analyzed for DNA morphology after addition of Hoechst 33342 DNA dye (not depicted). We observed condensed metaphase chromosomes at 75 min, and anaphase figures at 85 min after the start of the reaction.
	Figure 4. dnUbc9 does not alter decatenation activity of Xenopus topoisomerase II. (A) Chromosomal proteins were prepared from sperm chromatin assembled with or without 150 ng/μl dnUbc9 and assayed for decatenation activity as described in Materials and methods. Serial dilutions of chromosomal proteins were performed, in order to add the equivalent of the volumes indicated in each lane into decatenation reactions. For comparison, reactions were performed using purified human topoisomerase II, with the number of manufacturer's unit per reaction indicated. A fully decatenated marker was loaded in the lane marked D, and a linearized marker was loaded in the lane marked L. The lane marked 0 shows kinetoplast DNA from a control reaction without added chromosomal proteins. (B) Western blot of chromosomal eluates from extracts without (left) or with (right) dnUbc9 used in the decatenation reaction.
	Figure 5. SUMO-2/3–modified DNA topo-isomerase II is associated with chromatin. (A) CSF extracts containing 10,000 sperm nuclei/μl were incubated at 23°C for 60 min, and chromatin was isolated from each reaction as described in Materials and methods. Chromatin was extracted with XB buffer supplemented with 5 mM β-glycerol phosphate plus 100, 300, or 500 mM NaCl sequentially. Equal amounts of all fractions were subjected to SDS-PAGE and Western blotting with anti-topoisomerase II antibody, as indicated. (B) CSF extracts containing 10,000 sperm nuclei/μl were incubated at 23°C for 60 min, in the absence or presence of dnUbc9 (+dn; 150 ng/μl). Chromatin was isolated from each reaction as described in Materials and methods. Chromatin was extracted as above and equal concentrations of the supernatants from the 300-mM NaCl extraction (left two lanes in each panel), 500-mM NaCl extraction (middle two lanes in each panel), and final pellet (right two lanes in each panel) were subjected to SDS-PAGE and either silver staining (left) or Western blotting (right) with anti-topoisomerase II and anti-SUMO-2/3 antibodies. (C) Mitotic chromosomes were assembled in CSF extracts at 23°C for 60 min in the presence or absence of dnUbc9. The chromosomes were low salt (CSF-XB) or high salt (CSF-XB + 200 mM NaCl) buffers and stained with monoclonal anti–human topoisomerase IIα/β antibodies, as described in Materials and methods. DNA was visualized with Hoechst 33342. All topoisomerase II immunofluorescence images were taken using the identical exposure time.
	Figure 6. Perturbation of SUMO conjugation in mitosis causes chromosome segregation defects. (A) Schematic diagram of chromosome segregation assay. CSF extracts supplemented with rhodamine-labeled tubulin were released into interphase by CaCl2 addition. 2,000 sperm/μl were added 5 min after CaCl2, and the extract was incubated at 23°C for 55 min. Re-entry into mitosis was induced by addition of fresh CSF extract (50% of reaction volume) with or without dnUbc9 (150 ng/μl final concentration). After a 30 min incubation at 23°C, anaphase was induced with a second addition of CaCl2. (B) Aliquots were removed at the indicated times and analyzed for DNA morphology using Hoechst 33342 DNA dye (green) and for microtubule structures (red) after fixation. (C) The experiment was performed as above, with the addition of 35S-labeled securin at the time of the second aliquot of CSF extract. Samples at each time point were subjected to SDS-PAGE separation, and autoradiography using a Molecular Dynamics phosphorimager. (D) Mitotic chromatin were isolated from the reaction with or without dnUbc9 and subjected to Western blotting analysis with anti-SUMO2/3 and anti-topoisomerase II antibodies.

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