A temperature-sensitive DNA topoisomerase II mutant of the yeast Saccharomyces cerevisiae has been identified. Genetic analysis shows that a single recessive nuclear mutation is responsible for both temperature-sensitive growth and enzymatic activity. Thus, topoisomerase II is essential for viability and the mutation is most probably in the structural gene. Experiments with synchronized mutant cells show that at the nonpermissive temperature cells can undergo one, and only one, round of DNA replication. These cells are arrested at medial nuclear division. Analysis of 2-,um plasmid DNA from these cells shows it to be in the form of multiply intertwined catenated dimers. The results suggest that DNA topoisomerase II is necessary for the segregation of chromosomes at the termination of DNA replication.DNA topoisomerases are enzymes that catalyze the concerted breakage and rejoining of DNA backbone bonds (1). Topoisomerases can be divided into several categories, depending on their source and mode of action (reviewed in ref.2). Eukaryotic type 2 topoisomerases, the subject of this paper, can catalyze several different DNA isomerization reactions, including the relaxation, catenation, decatenation, knotting, and unknotting of closed double-stranded DNA circles (3-5). Although the eukaryotic type 2 DNA topoisomerases are fairly well characterized in vitro, nothing is known about their in vivo roles. It has been suggested that these enzymes might be involved in initiation of DNA replication (3) or in the segregation of daughter DNA molecules at the termination of DNA replication (6, 7).The properties of the yeast topoisomerases are quite similar to their counterparts from mammalian cells (8,9 MATa adel ade2 ura3-52, top2-1(ts), a segregant from the last backcross, was used for phenotype studies.Yeast Growth Conditions. Medium for growth of yeast was YPD (11) or YM-5 (12). The in vivo uniform labeling experiments were carried out essentially as described (12), except that [5,6-3H]uracil at 3 tCi/ml (41 Ci/mmol; 1 Ci = 37 GBq) was used. Cells were synchronized with a-factor pheromone as described (13). Nuclear staining was carried out as described (14), except that Hoechst 33258 dye was used instead of DAPI. Progress through the yeast cell cycle was monitored morphologically by phase-contrast microscopy (13).Topoisomerase Assays. Cells were grown in 25 ml of YPD medium at 250C. During exponential growth cultures were shifted to 370C for 20 min, chilled, centrifuged, washed with cold H20, and recentrifuged. The cell pellet was resuspended in 0.5 ml of yeast lysis buffer (20 mM Tris HCl, pH 7.5/1 mM Na2EDTA/1 mM dithiothreitol/1 mM phenylmethylsulfonyl fluoride/500 mM KCl/10% glycerol). Onethird volume of glass beads (Sigma, type IV,tm) was added and the cells were lysed by brief sonication. The lysate was centrifuged for 10 min in a desk top centrifuge.One microliter of the supernatant (undiluted or diluted in yeast lysis buffer plus 100 ,ug of bovine serum albumin per ml) was used for topoisomerase assays. D...