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.