Research Article 4763 Condensin-dependent localisation of topoisomerase II to an axial chromosomal structure is required for sister chromatid resolution during

Paula A. Coelho1, Joana Queiroz-Machado1,2 and Claudio E. Sunkel1,3,* 1Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal 2Faculdade de Ciências da Saúde, Universidade Fernando Pessoa, Praça 9 de Abril, 349, 4249-004 Porto, Portugal 3Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Porto, Portugal *Author for correspondence (e-mail: [email protected])

Accepted 24 July 2003 Journal of Cell Science 116, 4763-4776 © 2003 The Company of Biologists Ltd doi:10.1242/jcs.00799

Summary Assembly of compact mitotic and resolution significantly reduced topoisomerase II-dependent DNA of sister chromatids are two essential processes for the decatenation activity in vitro. Nevertheless, DmSMC4- correct segregation of the genome during mitosis. depleted chromosomes have centromeres and Condensin, a five-subunit protein complex, is thought to be that are able to segregate, although sister chromatid arms required for condensation. However, recent form extensive bridges during anaphase. These genetic analysis suggests that condensin is only essential to chromatin bridges do not result from inappropriate resolve sister chromatids. To study further the function of maintenance of sister chromatid cohesion by DRAD21, a condensin we have depleted DmSMC4, a subunit of the subunit of the complex. Moreover, depletion of complex, from Drosophila S2 cells by dsRNA-mediated DmSMC4 prevents premature sister chromatid separation, interference. Cells lacking DmSMC4 assemble short caused by removal of DRAD21, allowing cells to exit mitosis mitotic chromosomes with unresolved sister chromatids with chromatin bridges. Our results suggest that condensin where Barren, a non-SMC subunit of the complex is unable is required so that an axial chromatid structure can be to localise. Topoisomerase II, however, binds mitotic organised where topoisomerase II can effectively promote chromatin after depletion of DmSMC4 but it is no longer sister chromatid resolution. confined to a central axial structure and becomes diffusely distributed all over the chromatin. Furthermore, cell Key words: SMC4, DRAD21, Topoisomerase II, Condensin, extracts from DmSMC4 dsRNA-treated cells show Cohesin, Chromosomes, Mitosis, Drosophila

Introduction (Brn1p/Ycs3p/cnd2/XCAP-H/Barren/BRRN1, Ycg1/Yc5p/ During each cell division the genome of proliferating cells cnd3/XCAP-G/hCAP-G and Ycs4p/cnd1/XCAP-D2/hCAP- undergoes significant structural changes to ensure proper D2 in S. cerevisiae, S. pombe, C. elegans, D. melanogaster, segregation during mitosis. Chromosome condensation and Xenopus laevis and respectively), that are essential for resolution of sister chromatids is thought to be required to its activity both in vivo and in vitro (Cubizolles et al., 1998; resolve entangled chromatin fibres and to reduce chromosome Hirano et al., 1997; Kimura et al., 2001; Kimura and Hirano, arm length, as well as to increase the mechanical resistance of 2000; Lavoie et al., 2002; Lavoie et al., 2000; Ouspenski et al., chromatids for segregation. Condensation of chromatin into 2000; Sutani et al., 1999). Biochemical characterisation of the compact mitotic chromosomes is a highly complex process that complete condensin complex isolated from Xenopus extracts has only recently begun to be unravelled. A major (13S) showed that it is composed by two subcomplexes: an 8S breakthrough was the identification of condensin, a five- core subcomplex (8S), consisting of the SMC subunits XCAP- subunit protein complex (Hirano et al., 1997) that localises C/SMC4 and XCAP-E/SMC2 and an 11S regulatory throughout the axis of sister chromatids (Hirano et al., 1997; subcomplex (11S) containing the three non-SMC subunits Steffensen et al., 2001). Condensin is essential for cell viability XCAP-D2, XCAP-G, XCAP-H. The 11S complex is thought in , S. pombe, Caenorhabditis to play a regulatory role since it facilitates the ATP-dependent elegans, and humans (Biggins et al., positive supercoiling activity of the complex in vitro (Kimura 2001; Freeman et al., 2000; Hagstrom et al., 2002; Lavoie et et al., 2001). Even though both 8S and 13S can bind to DNA al., 2000; Saka et al., 1994; Steffensen et al., 2001; Strunnikov in vitro, only the 13S shows DNA-stimulated ATPase function et al., 1995; Sutani et al., 1999). Two of these subunits are and supports ATP-dependent chromatin condensation in SMC2 and SMC4 (DmSMC2 and DmSMC4 or gluon in Xenopus extracts (Kimura et al., 2001). Recently, in vitro Drosophila) that are members of the ubiquitous SMC studies in budding yeast showed that only the 13S complex is (structural maintenance of chromosomes) protein family. able to bind chromatin (Lavoie et al., 2002). The complex also contains three non-SMC proteins Chromosome condensation and sister chromatid segregation 4764 Journal of Cell Science 116 (23) also require topoisomerase II (Topo II) both in vivo (Uemura dissolution of sister chromatid cohesion in higher et al., 1987) and in vitro (Adachi et al., 1991; Hirano and occurs as a two-step process (for a review, see Losada and Mitchison, 1991). Topo II has been shown to be a major Hirano, 2001). During , as chromosomes condense, component of mitotic chromosomes where it localises to arm cohesion is lost through an APC/C-independent process. centromeres and throughout the axis of sister chromatids Subsequently, at anaphase onset, centromere-associated (Earnshaw et al., 1985; Gasser et al., 1986). More recent results cohesion is lost through an APC/C-dependent pathway show that in HeLa cells Topo II accumulates at discrete sites involving the Separase-mediated cleavage of the SCC1/Rad21 along chromatids alternating with sites where subunit. Barren localises (Maeshima and Laemmli, 2003). Since Since loss of arm cohesion occurs at the time when condensins have the ability to modify DNA Topology in vitro, chromosome condensation is taking place, it is possible that it is of great interest to determine how they interact with Topo the two processes are mechanistically linked. Indeed, it has II. Previously, it was shown that Barren interacts physically been shown in yeast that mutations in cohesion factors not only with Topo II and directly modulates its activity in vitro (Bhat affect sister chromatid cohesion but also perturb the et al., 1996). barren mutants exhibit chromosome segregation establishment and maintenance of chromosome condensation defects, in which the centromeres separate but chromosome (Castano et al., 1996). More recently, it has been demonstrated arms do not resolve. These mitotic phenotypes are very similar that proper function of condensins requires previous binding to those exhibited by DNA Topo II mutants of S. cerevisiae of to chromatin (Lavoie et al., 2002). However, in (Holm et al., 1985) with the exception that they do not higher eukaryotes the loss of cohesins does not affect accumulate catenated DNA molecules or show chromosome chromosome condensation (Losada et al., 1998; Sonoda et al., breakage (Bhat et al., 1996). However, we have recently shown 2001; Vass et al., 2003). that Drosophila cells mutant for dmSmc4 show a phenotype In this study we analysed the role of condensin in the reminiscent of yeast Topo II mutants (Steffensen et al., 2001). organisation and segregation of mitotic chromosomes by Loss of DmSMC4 in Drosophila allows normal longitudinal depleting DmSMC4 in Drosophila S2 cells. We show that shortening of the chromosome but sister chromatids are not DmSMC4 can be depleted in S2 cells by dsRNAi resulting resolved resulting in the formation of anaphase chromatin in abnormal chromosome condensation and segregation. bridges and DNA breakage. Since Topo II catalytic activity is Depletion of DmSMC4 also affects the localisation of required for the contraction of chromosomes (Gimenez-Abian Barren, a non-SMC subunit of the condensin complex, to et al., 1995; Gorbsky, 1994), it is likely that in the absence of mitotic chromatin, suggesting that only the complete DmSMC4, Topo II remains functional. However, Topo II also condensin complex binds chromatin in vivo. Furthermore, mediates decatenation of DNA early in mitosis (Gimenez- after depletion of DmSMC4 Topo II fails to localise to a Abian et al., 1995), which might be affected in the Drosophila clearly defined chromatid axial structure and protein extracts DmSmc4 mutant resulting in the formation of chromatin from DmSMC4-depleted cells exhibit a significant decrease bridges during anaphase. in Topo II DNA decatenation activity. Surprisingly, However, anaphase bridges could also result from abnormally condensed chromosomes in DmSMC4-depleted inappropriate maintenance of cohesion between sister cells appear to organise functional kinetochores that bind chromatid arms. Cohesion of sister chromatids during most of microtubules and undergo segregation. We also show that the is mediated by cohesin, a protein complex made depletion of DmSMC4 does not affect the localisation or up of at least four subunits including SMC1 and SMC3, two removal of the cohesin complex during mitosis suggesting members of the SMC protein family, and two non-SMC that the chromatin bridges observed after depletion of subunits, Scc1p/Mcd1p (Rad21p in fission yeast and DRAD21 condensin are not due to inappropriate localisation of in Drosophila) and Scc3p (Guacci et al., 1997; Michaelis et cohesin. However, depletion of DmSMC4 prevents al., 1997; Toth et al., 1999). In yeast, cohesion is established premature sister chromatid separation caused by loss of during S phase and cohesins remain tightly associated with DRAD21, and allows cells to exit mitosis. Our results chromosomes until the -anaphase transition, when suggest that condensin is required for the organisation of a Scc1p is cleaved in a reaction dependent on a cysteine protease central axial chromatin structure where Topo II can localise called Separase (Nasmyth et al., 2000; Uhlmann et al., 1999). and promote sister chromatid resolution. This transition is triggered by the activation of the anaphase- promoting complex/cyclosome (APC/C) (Ciosk et al., 1998; Cohen-Fix et al., 1996; Funabiki et al., 1996; Glotzer, 1999; Materials and Methods Uhlmann et al., 1999), which targets a protein called RNA interference Securin for degradation by ubiquitin-dependent proteolysis. RNAi was performed in Drosophila S2 tissue culture cells as Destruction of Securin releases Separase, which can now previously described (Clemens et al., 2000). A 950 bp EcoRI-SalI or cleave Scc1p. In Xenopus egg extracts (Losada et al., 1998), an 800 bp EcoRI-KpnI fragment from the 5′ end of DmSMC4 or (Sumara et al., 2000) and mouse cells (Darwiche et al., DRAD21 cDNAs, respectively, were cloned into both pSPT18 and 1999) cohesin binds to chromatin during but pSPT19 expression vectors (Roche). The recombinant plasmids were dissociates from chromosome arms early in mitosis, while used as templates for RNA synthesis using the T7 Megascript kit (Ambion) and 15 µg of dsRNA were added to 106 cells in all RNAi centromere-associated cohesin remains bound until the experiments. At each time point, cells were collected and processed metaphase-anaphase transition where it holds sister chromatids for immunoblotting or immunofluorescence. For immunoblotting, 106 together until anaphase onset (Hoque and Ishikawa, 2001; cells were collected by centrifugation, resuspended in 20 µl of sample Losada et al., 2000; Waizenegger et al., 2000; Warren et al., buffer, sonicated, and boiled for 3 minutes before loading on 5-20% 2000). These observations have led to a model in which gradient SDS-polyacrylamide gels. The role of condensins in mitosis 4765

Immunofluorescence in S2 cells supernatant was adjusted to 70% (NH4)2SO4 with a saturated solution S2 cells were fixed in 3.7% formaldehyde, 0.5% Triton X-100 in PBS and left at 4°C for 30 minutes. The precipitated proteins were for 10 minutes and then washed three times for 5 minutes in PBS-T collected by centrifugation at 10,000 g for 15 minutes at 4°C and (PBS with 0.05% Tween 20). Blocking was performed in PBS-TF resuspended in 0.5 ml dialysis buffer (40 mM Tris-HCl, pH 7.5, 2 mM (PBS-T with 10% fetal calf serum) for 1 hour at room temperature. DTT, 1 mM EDTA, 0.25 M sucrose). Incubations with primary antibody were performed in PBS-TF overnight at 4°C or for 2 hours at room temperature. Slides were washed again three times for 5 minutes in PBS-T. Incubation of Topoisomerase II assays fluorescently labelled secondary antibodies was done according to Topo II assays were performed with kinetoplast DNA (kDNA) manufacturer’s instructions (Jackson ImmunoResearch Laboratories extracted from cultures of Leishmania infantum clone and Vector Labs, UK). Slides were washed with PBS-T (3 times MHOM/MA67ITMAP263 as previously described (Kelly, 1993) in 5 minutes each), and then mounted in Vectashield (Vector, UK) the presence of 1 mM ATP. kDNA decatenation assays were containing either 1 µg/ml of DAPI, for observation in the Zeiss performed for 30 minutes at 37°C and stopped by addition of 4 µl of Axiovert 200M microscope (Zeiss, Germany), or 1.0 µg/ml propidium 2.5% SDS; 25 mM EDTA, 30% sucrose, 0.25% Bromophenol Blue. iodide for confocal microscopy, using a Biorad MRC600 microscope (Biorad, UK). Image stacks were recorded with an Axiocam (Carl Zeiss, Germany). Data stacks were deconvolved, using the microscope Results Axiovision 3.1 Software (Carl Zeiss, Germany). Depletion of SMC4 by dsRNA interference in S2 cells In order to deplete DmSMC4, Drosophila S2 cells were Antibodies treated with dsRNA and the levels of the protein was The primary antibodies were anti-α-tubulin (mouse mAb B512), monitored at different times by immunoblotting (Fig. 1A). used at 1:3,000 (Sigma-Aldrich); anti-phosphorylated H3 rabbit DmSMC4 protein decreased significantly at 48 hours and was polyclonal, used at 1:500 (Upstate Biotechnology); anti-Cid chicken barely detectable 120 hours after treatment. Titration of the polyclonal, used at 1:200 (Blower and Karpen, 2001); anti-DmSMC4 antibody confirmed that at these cell densities the protein antibodies (rabbit, 1:500 or sheep, 1:200) as described previously should have been easily detected (Fig. 1B). Cell viability was (Steffensen et al., 2001); anti-DRAD21 (Warren et al., 2000) rabbit not affected as determined by Trypan Blue staining (Fig. 1C) polyclonal (1:500); anti-Barren rabbit polyclonal, used at 1:1000 and the doubling time of control and DmSMC4 dsRNA- (Bhat et al., 1996), anti-Polo mouse mAb MA294, used at 1:50 treated cells was similar throughout the experiment (Fig. 1D). (Llamazares et al., 1991) and anti-Topo II mouse monoclonal antibody P2G3 used at 1:20 (Swedlow et al., 1993). The percentage of mitotic cells and overall phenotype of DmSMC4 dsRNA-treated cells was analysed after immunostaining with an anti-phophohistone H3 antibody to Mitotic chromatin immunoprecipitation assay identify mitotic cells (Fig. 1E-G; Table 1). The mitotic index Mitotic chromatin immunoprecipitation assays were carried out as in DmSMC4-depleted cells was similar to that of controls described previously (Mirkovitch et al., 1988). S2 cells were grown (Fig. 1E) and mitotic progression was not significantly exponentially to 2-6×106 cell/ml, blocked in mitosis with 30 µM affected (Fig. 1F). However, soon after DmSMC4 RNAi colchicine for 8 hours, incubated on ice for 45 minutes and treatment (24 hours) mitotic cells had abnormally condensed centrifuged at 1500 g for 15 minutes at 4°C. Cells were resuspended chromosomes that did not show well-defined sister in 1 ml of cold PBS in the presence of protease inhibitors, kept on ice chromatids, and chromatin bridges were observed in anaphase for 45 minutes and lysed in 1 ml of lysis buffer (15 mM Tris-HCl, pH 7.4, 0.2 mM spermine, 0.5 mM spermidine, 2 mM K-EDTA, 1 mM cells (Fig. 1G and Table 1; see also Fig. 2). At later times EGTA, 150 mM KCl, 15 mM NaCl, 1 mM DTT, 0.05% Triton X- virtually all mitotic cells showed abnormal chromosome 100) with 0.1% (v/v) digitonin. Cells were lysed with a B-type pestle condensation during prometaphase and chromatin bridges in a Dounce homogeniser. Dyna-beads coated with sheep anti-rabbit during anaphase and telophase (Table 1). Incubation of IgG (Dynal Biotech) were washed with lysis buffer. 5 µl of Dyna- DmSMC4-depleted cells with colchicine causes mitotic arrest beads were used to clear 1 ml of cell protein extract. Mitotic chromatin with unseparated sister chromatids indicating that the spindle was labelled by incubating cell extracts with anti-phosphohistone H3 (Upstate Biotechnology) overnight at 4°C. Immune complexes were captured by incubation with 10 µl of Dyna-beads (6-7×107 beads/µl) Table 1. Quantification of mitotic phenotypes at different according to the manufacture’s instructions. Complexes were washed times after depletion of DmSMC4 by RNAi 3 times with lysis buffer and resupended in 50 µl of 2× Laemmli Chromatin bridges sample buffer. *Unresolved sister chromatids (%) Anaphase (%) Telophase (%) Preparation of cell extracts Time (h) Control RNAi Control RNAi Control RNAi The preparation of cell extracts for Topo II assays was done using 30 24 0 28.6 0 0 0 6.3 ml of exponentially growing cells at 2-6×106 cell/ml as previously 48 0 54.5 0 25.0 0 4.0 described (Mohamed et al., 1987). Cells were incubated on ice for 45 72 0 73.7 0 46.2 0 50.1 96 0 97.2 0 61.5 0 73.7 minutes, collected by centrifugation at 1500 g for 15 minutes at 4°C 120 0 100 0 50.0 0 89.5 and resuspended in 1 ml of cold PBS. Cells were then centrifuged for 144 0 100 0 85.7 0 94.4 10 minutes at 4°C and washed in 5 ml of extraction buffer (50 mM Tris-HCl pH 7.5, 25 mM KCl, 3 mM MgCl2, 0.25 M sucrose, 2 mM *Cells in prometaphase. Total number of cells counted for controls: 24 DTT, 1 mM EDTA) in the presence of proteinase inhibitors. Pellets hours n=1768, 48 hours n=2041, 72 hours n=2304, 96 hours n=2724, 120 were resuspended in 2 ml of the same buffer and homogenised in a hours n=2066 and 144 hours n=1146. Total number of cells counted for Dounce homogeniser at 4°C. The extracts were adjusted to 400 mM RNAi-treated cells: 24 hours n=1792, 48 hours n=1958, 72 hours n=2270, 96 KCl and centrifuged at 30,000 rpm for 60 minutes at 4°C. The hours n=2888, 120 hours n=1902 and 144 hours n=2091. 4766 Journal of Cell Science 116 (23)

Fig. 1. Depletion of DmSMC4 in Drosophila S2 cells by dsRNAi. (A) Depletion of DmSMC4 was monitored over time by western blot analysis. Total protein extracts from 106 cells were separated by SDS-PAGE and immunoblotted with anti-DmSMC4 or α-tubulin as a loading control. Quantification of depletion was determined by normalising for α- tubulin. (B) Protein extracts of different concentration of control cells were immunoblotted with anti-DmSMC4 antibody to determine the level of antibody detection. (C) Viability index of control and DmSMC4 dsRNAi- treated cells was determined by Trypan Blue staining. (D) Proliferation of control or DmSMC4 dsRNAi-treated S2 cells was monitored throughout the experiment. (E) Quantification of the mitotic index during the course of the experiment in control cells and cells treated with DmSMC4 dsRNAi was determined by counting phosphohistone H3-positive cells. (F) Anti- phosphohistone H3-positive cells were also used to quantify mitotic progression in control (C) and RNAi (R)-treated cells. Numbers of cells counted for D and E is shown in Table 1. (G) Phenotypic analysis of phosphohistone H3 positive cells in control and DmSMC4 dsRNAi-treated cells. After depletion of DmSMC4, phosphohistone H3 staining persists on chromatin bridges during telophase (arrow). Representative images of metaphase chromosomes at a higher magnification are shown in the insets. Note that sister chromatids do not resolve after DmSMC4 depletion. Scale bars: 5 µm. checkpoint is active in these cells (data not shown: see also we analysed the distribution of Barren on mitotic chromatin Fig. 3B). These results indicate that depletion of DmSMC4 after depletion of DmSMC4 (Fig. 2). In control prometaphase does not compromise the length-wise compaction of mitotic and anaphase cells, DmSMC4 and Barren showed co- chromosomes but causes abnormalities in sister chromatid localisation to a well-defined chromosomal axial structure. By resolution and segregation in Drosophila S2 cells. contrast, soon after the addition of DmSMC4 dsRNA, when cells showed a significant reduction in the level of DmSMC4, chromosomes displayed abnormal condensation and most did Immunolocalisation of Barren after depletion of not resolve sister chromatids. More significantly, the lower DmSMC4 levels of DmSMC4 or Barren detected in these chromosomes In order to evaluate whether DmSMC4 is required for the no longer localised to a well-defined chromatin axis but localisation of non-SMC proteins of the condensin complex, appeared diffusely distributed all over the chromatin. After The role of condensins in mitosis 4767

Fig. 2. Immunolocalisation of DmSMC4 and Barren in control and DmSMC4- depleted Drosophila S2 cells. In control cells during prometaphase and anaphase DmSMC4 and Barren co-localise to an axial structure of sister chromatids. After depletion of DmSMC4 by dsRNAi, chromosomal-associated DmSMC4 is significantly reduced after 48 hours and undetectable after 96 h. A reduction in the level of chromatin-associated DmSMC4 causes the formation of short chromosomes with unseparated sister chromatids and a highly abnormal distribution of Barren. In cells that show no DmSMC4 staining, chromosomes are short, no defined sister chromatids are observed and Barren is undetected. Scale bars: 5 µm.

Organisation of centromeres and kinetochores in the absence of DmSMC4 DmSMC4-depleted cells show abnormally condensed chromosomes, however these cells progress into anaphase and attempt to segregate their chromosomes but display extensive chromatin bridges. To determine whether centromere and organisation is normal, cells lacking DmSMC4 were immunostained for a centromere marker CID, the Drosophila homologue of CENP-A (Henikoff et al., 2000) and for Polo, a protein that localises to the kinetochore (Logarinho and Sunkel, 1998). In control cells CID localised to the centromeres of mitotic chromosomes and lead sister chromatids during segregation (Fig. 3A). Depletion of DmSMC4 did not alter the localisation of CID, which appeared as discrete dots over the abnormally condensed chromatin throughout mitosis (Fig. 3B). However, in early anaphase cells, centromeres as shown depletion of DmSMC4 for longer periods the protein was not by CID staining, appeared to stretch poleward sometimes well detected by immunofluorescence and Barren was completely beyond the chromatin and during later stages centromeres were absent from the abnormally condensed chromosomes. All clearly leading sister chromatids to the poles. Dots of CID prometaphase cells contained chromosomes with unresolved staining were only rarely observed associated with chromatin sister chromatids and most cells in anaphase/telophase showed bridges during anaphase. If cells were incubated with chromatin bridges (Fig. 2). Since Barren was detected in colchicine to depolymerise microtubules and thus prevent exit DmSMC4-depleted cell extracts by immunoblotting (see from mitosis, CID staining was consistently observed as two below and data not shown), these results indicate that its dots on each side of prometaphase chromosomes as in control localisation to mitotic chromatin requires DmSMC4. These cells (Fig. 3B). findings suggest that only an intact condensin complex is able To analyse kinetochore organisation DmSMC4-depleted to associate with chromatin and that normal levels of DmSMC4 cells were immunostained for Polo (Fig. 3C,D). In control cells are needed to maintain its localisation to a clearly defined Polo localised to the centrosomes from late G2 to cytokinesis chromatid axis. and to kinetochores from early prometaphase to late anaphase 4768 Journal of Cell Science 116 (23)

Fig. 3. Analysis of centromere and kinetochore function after depletion of DmSMC4. (A) Immunofluorescence analysis of CID in control cells shows discrete localisation to centromeres in prometaphase and anaphase. (B) Localisation of CID in DmSMC4-depleted cells shows centromere staining throughout mitosis. In early anaphase cells, CID staining appears to extend polewards beyond the mitotic chromatin (arrows) and in late anaphase centromeres lead migrating chromatids to the spindle poles. After incubation with colchicine, CID-stained centromeres localise over the chromosomes. (C) Control cell immunostained with anti-POLO antibodies show localisation to kinetochores and to centrosomes located at either side of the metaphase plate. (D) After depletion of DmSMC4, POLO localises to kinetochores that during anaphase can also be seen to stretch towards the poles. (E) Control cells stained for CID and tubulin show centromeres associated with microtubule bundles. A higher magnification image is shown in the left panel. (F) In DmSMC4-depleted cells centromeres stretched beyond the chromatin associated with microtubule bundles. Higher magnification images are shown in the left panels. Scale bars: 5 µm.

(Fig. 3C). In DmSMC4-depleted cells (Fig. 3D), Polo 3F). These results suggest that in the absence of DmSMC4 localisation at kinetochores showed a very similar pattern to centromere and kinetochore components localise properly, and that described for CID. During early anaphase, kinetochores that kinetochores bind microtubules and are mostly segregated stained for Polo also appeared to stretch poleward outside the to the poles of the spindle. Furthermore, our data also chromatin. This phenotype was not observed after incubation shows that during early stages of anaphase centromeres and with colchicine (data not shown). To determine whether these kinetochores are significantly stretched towards the poles kinetochores were able to bind to microtubules, DmSMC4- suggesting that centromeric cohesion is likely to be resolved depleted cells were immunostained for CID, tubulin and DNA but sister chromatids arms are unable to segregate properly. (Fig. 3E,F). In control cells, microtubule bundles can be clearly seen to run from the spindle poles and terminate at CID- positive centromeres. In DmSMC4-depleted cells, microtubule Immunolocalisation of Topo II in the absence of bundles were also observed to contact CID-positive DmSMC4 centromeres that appear to be located outside chromatin (Fig. Absence of DmSMC4 in Drosophila results in the formation The role of condensins in mitosis 4769

Fig. 4. Localisation of Topo II to mitotic chromatin after depletion of DmSMC4. (A) In control cells DmSMC4 and Topo II partially co-localise to an axial structure of sister chromatids during both prometaphase or anaphase. Some accumulation of Topo II is also observed at the centromeres. Merged images show DNA (red) and Topo II (green). (B) Detailed analysis of distribution of DmSMC4 and Topo II in prometaphase chromosomes. The merged image shows DmSMC4 (green) and Topo II (red). Higher magnification view of selected regions (a and b) indicated in the merged image, show that Topo II and DmSMC4 localise mostly as an alternating string pattern with some regions in which both proteins are found. (C) After depletion of DmSMC4, Topo II does not show a defined localisation to the axial structure within sister chromatids but becomes diffusely distributed all over condensed chromatin and in chromatin bridges. Merged images show DNA (red) and Topo II (green). Scale bars: 5 µm.

chromatids and stronger accumulation at the centromeric region was sometimes observed (Fig. 4A). However, detailed analysis of prometaphase chromosomes stained for both DmSMC4 and Topo II showed that there is only partial co-localisation (Fig. 4B). The localisation of DmSMC4 and Topo II appears to alternate along the axis of sister chromatids with some regions of overlapping staining. After depletion of DmSMC4, the localisation of Topo II was significantly different (Fig. 4C). In all prometaphase and metaphase cells analysed, Topo II was now found diffusely distributed all over the chromosomes and a defined central axis was never observed. In cells undergoing anaphase, Topo II was also diffusely distributed on the chromatin bridges (Fig. 4C). In order to determine whether loss of DmSMC4 caused a reduction in the level of Topo II associated with mitotic chromatin we carried out chromatin immunoprecipitation assays followed of anaphase chromatin bridges, a phenotype that was also by immunoblotting (Fig. 5). In total protein extracts we found described for temperature-sensitive top2 mutations in yeast that the level of Topo II was reduced (19.1%) as compared to (DiNardo et al., 1984; Uemura et al., 1987). It has been controls (Fig. 5A). This reduction in the level of Topo II suggested that condensin might regulate Topo II-mediated appears to be associated only with the non-mitotic decatenation of sister chromatids in vivo (Hirano, 2000; chromosome fraction since immunoblotting of mitotic Koshland and Strunnikov, 1996). Therefore, we next asked chromatin immunoprecipitated by anti-phosphohistone H3 whether DmSMC4 was required for the proper localisation of antibodies showed significant Topo II levels (Fig. 5B). These Topo II (Fig. 4). In control mitotic cells, Topo II mostly co- results suggest that DmSMC4 is not required for the localised with DmSMC4 throughout the axis of sister association of Topo II to mitotic chromatin but is required for 4770 Journal of Cell Science 116 (23)

Fig. 5. Determination of the level of Topo II associated with chromatin in DmSMC4- depleted cells. (A) The levels of Topo II in total protein extracts from control or DmSMC4- depleted cells after 96 hours show a 19.5% reduction. (B) To determine whether this reduction corresponds to the chromosomal-associated Topo II, increasing amounts of mitotic chromatin extracts from control and DmSMC4-depleted cells immunoprecipitated with anti- phosphohistone H3 antibodies or the remaining supernatant were separated by SDS-PAGE and immunoblotted. Membranes were probed with antibodies against DmSMC4 and Topo II. H3 was used as a loading control for immunoprecipitated chromatin and α-tubulin for the supernatant. While DmSMC4 and Topo II are clearly found in the control extracts, in the chromatin from DmSMC4-depleted cells DmSCM4 is absent and the levels of Topo II are not significantly different from controls extracts. In the corresponding supernatant fractions, DmSMC4 and Topo II are easily detected in the control cells. However, after DmSMC4 dsRNAi treatment, DmSMC4 is not detected and Topo II is reduced. the localisation of Topo II to a defined central axial structure not accumulate at a defined axial structure within sister where it is normally confined. Furthermore, the data suggest chromatids. To determine whether depletion of DmSMC4 that depletion of DmSMC4 causes a reduction in the levels of might have an effect upon the DNA decatenation activity of non-chromosomal associated Topo II. Topo II, we devised an in vitro assay. Protein extracts from either control or DmSMC4 dsRNAi-treated cells were prepared and kDNA decatenation activity of Topo II (Marini et al., 1980) Topo II decatenation activity in the absence of DmSMC4 was tested (Fig. 6). The results showed that extracts from The results presented above indicated that after depletion of control cells (Fig. 6A) were able to induce decatenation of the DmSMC4, Topo II can associate with chromatin but does kDNA and addition of the specific inhibitor ICRF-187 (Roca et al., 1994) completely abolished this activity. However, when DmSMC4-depleted cell extracts were used (Fig. 6B), no significant amount of decatenated DNA was detected. If exogenous Topo II was added in excess, DNA decatenation was observed, indicating that the cell extracts are capable of supporting this activity. These results suggest that the endogenous Topo II appears to be dependent upon the presence of DmSMC4 for its decatenation activity. The interaction

Fig. 6. In vitro activity of Topo II in control and DmSMC4-depleted extracts. (A) Determination of Topo II activity of chromatin- associated protein extracts from control cells using kDNA as substrate. For each assay 10 µg of total extracts with (+) and without (–) 10 µM of Topo II inhibitor ICRF-187 were used and the reaction was allowed to proceed for different times. The DNA was deproteinised and analysed on a 0.8% agarose gel. In control extracts the kDNA was rapidly decatenated to produce relaxed minicircles after 5 minutes of the incubation. The addition of ICRF-187 for 10 minutes completely inhibited decatenation. (B) Decatenation of kDNA was used to measure Topo II activity in control or DmSMC4- depleted extracts after 96 hours of treatment without (–) or with (+) the addition of exogenous Topo II (2U, Amersham). Different levels of protein extracts were used: 0 µg (−), 5 µg (+) or 10 µg (++). DNA was deproteinised and run on a 0.8% agarose gel containing 0.5 µg/ml ethidium bromide. Decatenation of kDNA is observed in control extracts with or without addition of exogenous Topo II. DmSMC4-depleted extracts showed no decatenation activity in the absence of exogenously added Topo II. The role of condensins in mitosis 4771

Fig. 7. Localisation of DRAD21 in DmSMC4- depleted Drosophila S2 cells. (A) Immunolocalisation of DmSMC4 and DRAD21 in control cells. In prometaphase cells, DmSMC4 and DRAD21 do not appear to co- localise. DRAD21 is always confined to the centromeric region between the axial structure stained by DmSMC4. During anaphase while DmSMC4 localises throughout sister chromatids, DRAD21 is not detected. (B) After depletion of DmSMC4, DRAD21 is still highly abundant in prophase chromosomes but during prometaphase it is restricted to defined regions of the condensed chromosomes. In anaphase, DRAD21 is never detected either on the chromatids or the chromatin bridges. Scale bars: 5 µm. (C) Western blot of total protein extracts from control and DmSMC4- depleted cells after 96 hours show that depletion of DmSMC4 does not affect the overall levels of DRAD21 but Barren levels are reduced compared to control extracts. (D) Analysis of protein extracts from immunoprecipitated mitotic chromatin of either control or DmSMC4-depleted cells and the corresponding supernatants. In chromatin extracts from control cells DmSMC4, DRAD21 and Barren can be easily detected while in extracts from dsRNAi-treated cells neither DmSMC4 nor Barren are detected and the level of DRAD21 is reduced compared to controls. Phosphohistone H3 was used as a loading control. The corresponding supernatants are shown below. DmSMC4 is not detected by immunoblotting and the levels of DRAD21 are not different from controls. However, note that depletion of DmSMC4 causes a significant decrease in the level of Barren present in the supernatant fraction. α-tubulin was used as loading control.

between Topo II and SMC4 is unlikely to be direct since we were unable to detect a specific interaction by immunoprecipitation (data not shown).

Immunolocalisation of cohesin after depletion of DmSMC4 DmSMC4-depleted mitotic chromosomes are unable to localise Topo II to a defined axial structure and its in vitro activity appears to be compromised, suggesting that sister chromatid resolution requires the concerted activity of condensin and Topo II. However, since an interaction between condensins and cohesins has been suggested, it is also possible that loss of DmSMC4 could lead to inappropriate maintenance of cohesin at chromosome arms during mitotic exit resulting in the formation of chromatin bridges. Therefore, we analysed the distribution of DRAD21 (the Drosophila homologue of the SCC1 cohesin subunit) in DmSMC4-depleted cells (Fig. 7). In control cells (Fig. 7A) DmSMC4 was confined to the 4772 Journal of Cell Science 116 (23) axis of sister chromatids while DRAD21 localised to the slightly elevated mitotic index (Fig. 8E) with highly condensed centromeric and pericentromeric regions between the axis of and separated sister chromatids (82.6%). Analysis of mitotic the two sister chromatid, showing that DmSMC4 and DRAD21 progression (Fig. 8F) indicated that DRAD21-depleted cells never co-localise. During anaphase, DmSMC4 was detected accumulated mostly in prometaphase (68.9%) and some along sister chromatid arms and DRAD21 was no longer displayed an anaphase-like configuration (3.5%). Most present. In DmSMC4-depleted cells (Fig. 7B) DRAD21 importantly, no cells with chromatin bridges during anaphase localised to chromatin at prophase but during prometaphase or telophase were observed. These results are in agreement was present only at pericentromeric and centromeric regions of with previous reports (Sonoda et al., 2001; Vass et al., 2003). the abnormally condensed chromosomes. More significantly, We then carried out simultaneous depletion of DmSMC4 and during anaphase or telophase, DRAD21 was never found DRAD21, and after 96 hours the level of both proteins was localised to the chromatin bridges. These results suggest that significantly reduced (Fig. 8B). Surprisingly, mitotic cells cohesin does not appear to be responsible for the formation of showed a phenotype very different from that resulting from chromatin bridges after anaphase onset since its release from depletion of DRAD21 alone (Fig. 8D). Cells depleted of mitotic chromatin in DmSMC4-depleted cells follows normal DRAD21 and DmSMC4 showed abnormal chromosome kinetics. condensation with unresolved sister chromatids (86.4%), very In order to confirm these results, we analysed the levels of low levels of premature sister chromatid separation (3.3%) cytoplasmic or mitotic chromatin-associated DRAD21 from and all anaphase or telophases had chromatin bridges. either control or DmSMC4 dsRNA-treated cells. Western blots Quantification of mitotic progression (Fig. 8F) showed that of total protein extracts from control and DmSMC4-depleted cells depleted of both DRAD21 and DmSMC4 progress cells show that DmSMC4 is significantly reduced, DRAD21 is through prophase and prometaphase but accumulate in not affected while and the level of Barren is also reduced anaphase and telophase. These cells are clearly exiting mitosis as compared to α-tubulin, the loading control (Fig. 7C). since cyclin B levels are not different from those of control Mitotic chromatin was then immunoprecipitated with anti- cells at similar mitotic stages (data not shown). These phosphohistone H3 antibodies, separated by SDS-PAGE and observations support the results of the previous section and immunoblotted against DmSMC4, DRAD21, Barren and show that chromatin bridges present in DmSMC4-depleted Histone H3 or α-tubulin as loading controls (Fig. 7D). cells are formed independently of DRAD21. Furthermore, the Increasing amounts of the immunoprecipitates for either data shows that depleting DmSMC4 prevents premature sister non-treated or DmSMC4 dsRNA-treated cells were used. chromatid separation and overrides the prometaphase arrest DmSMC4, DRAD21 and Barren were easily detected in the caused by loss of DRAD21. immunoprecipitated chromatin fraction from non-treated cells. However, in mitotic chromatin immunoprecipitated from DmSMC4-depleted cells, neither DmSMC4 nor Barren could Discussion be detected. More significantly, DRAD21 not only did not We have shown that dsRNAi can be used to severely deplete accumulate to higher levels but was somewhat reduced as DmSMC4 in tissue culture cells resulting in mitotic compared to controls. Analysis of the supernatant fraction phenotypes that are very similar to those previously described showed that DmSMC4 was not present, as expected, while for dmSmc4 mutant Drosophila cells (Steffensen et al., 2001). DRAD21 was present at significant levels. Also, we observed Already 24 hours after RNAi treatment some mitotic cells that although Barren was still detected in the extracts from show abnormal resolution of sister chromatids and later, cells DmSMC4-depleted cells, its levels in the non-chromatin in anaphase or telophase begin to show chromatin bridges fraction were consistently decreased. These results suggest indicating that the frequency with which these phenotypes are that the distribution of cohesins and their release from observed depends on the level of depletion of DmSMC4. chromosomes during mitotic progression is not affected by the Loss of DmSMC4 causes the formation of short mitotic depletion of DmSMC4. chromosomes with poorly defined sister chromatids. These chromosomes are unlikely to contain other proteins of the condensin complex since immunofluorescence and mitotic Mitotic progression after simultaneous depletion of chromatin immunoprecipitation shows that binding of Barren DRAD21 and DmSMC4 to condensing chromosomes is dependent on DmSMC4. These To exclude the possibility that chromatin bridges, observed observations are in agreement with previous findings indicating after depletion of DmSMC4, might be due to some DRAD21- that non-SMC condensins can only bind DNA in the presence dependent mechanism we carried out simultaneous depletion of the entire condensin complex (Cubizolles et al., 1998; of DmSMC4 and DRAD21. The results showed that chromatin Hirano et al., 1997; Kimura et al., 2001; Kimura and Hirano, bridges can form independently of DRAD21 (Fig. 8). Firstly, 2000; Lavoie et al., 2000; Ouspenski et al., 2000; Sutani et al., we depleted DRAD21 alone. Titration of the anti-DRAD21 1999). Also, it was shown in S. pombe and S. cerevisiae that antibody showed that the number of cells used at each time all members of the regulatory subcomplex are essential for point was sufficient to easily detect the protein (Fig. 8A) and chromatin association of yeast condensin in vivo (Freeman et that the level the protein was not affected in control cultures al., 2000; Lavoie et al., 2002). Together, these results strongly (Fig. 8B). However, cells treated with DRAD21 dsRNA suggest that, in Drosophila, the assembly of the condensin showed significant depletion of the protein by 96 hours (Fig. complex to mitotic chromatin requires all protein subunits. 8B). To determine whether depletion of DRAD21 causes Moreover, our results demonstrate that certain aspects mitotic abnormalities, cells were stained for DRAD21 and of chromosome condensation, namely shortening of the DNA (Fig. 8C). Cells treated with DRAD21 RNAi showed a longitudinal axis of sister chromatids, can occur in the absence The role of condensins in mitosis 4773

Fig. 8. Simultaneous depletion of DmSMC4 and DRAD21 in Drosophila S2 cells. (A) Protein extracts from different concentrations of cells were immunoblotted with anti-DRAD21 antibody to determine the level of detection of the antibody. (B) Protein extracts from control or DRAD21-depleted cells were immunoblotted to detect DRAD21, DmSMC4 and α-tubulin as a loading control. Note that significant depletion of DRAD21 is obtained 72 hours after treatment with RNAi while the levels of DmSMC4 remain unchanged. Cells were also treated with dsRNA for DRAD21 and DmSMC4 simultaneously and protein extracts prepared at different times and immunoblotted to reveal DRAD21, DmSMC4 and α- tubulin as loading control. Significant depletion of both proteins was obtained after 96 hours of treatment therefore all phenotypic and quantification analysis was carried out at this time. (C) Cells depleted of DRAD21, stained for DRAD21 and DNA, show premature sister chromatid separation and highly condensed chromatids. (D) Cells depleted of both DmSMC4 and DRAD21, stained for DRAD21, DmSMC4 and DNA, show abnormal chromosome condensation and poorly defined sister chromatids during prometaphase and during anaphases have extensive chromatin bridges. (E) Quantification of mitotic index of control cells and cultures treated with different dsRNAs shows some accumulation of mitotic cells after depletion of DRAD21 or simultaneous depletion of DRAD21 and DmSMC4. (F) Quantification of mitotic progression after treatment of cells with different dsRNAs. Depletion of DmSMC4 does not affect mitotic progression while depletion of DRAD21 causes a significant prometaphase arrest with few cells in metaphase or anaphase. Simultaneous depletion of DmSMC4 and DRAD21 causes a significant accumulation of cells in anaphase and telophase. The number of cells used for the quantifications shown in E and F was: control n=1148, DmSMC4 RNAi n=1038, DRAD21 RNAi n=1923 and simultaneous DmSMC4/DRAD21 RNAi (double) n=1801. Scale bars: 5 µm. of condensins, fully supporting our previous results (Steffensen observed that at early stages of mitosis, kinetochores et al., 2001). associated with spindle microtubules appear to stretch Although, chromosomes do not condense normally in poleward, sometimes well beyond the chromatin. However, DmSMC4-depleted cells, genetic studies in Drosophila when microtubules are depolymerised by colchicine, showed that loss of DmSMC4 (Steffensen et al., 2001) or kinetochores localise only over mitotic chromatin, suggesting Barren (Bhat et al., 1996) does not prevent cells from entering that stretching of kinetochores is microtubule dependent. anaphase and attempting sister chromatid segregation. Similar observations were reported after expressing GFP- However, recently it has been suggested that the condensin tagged centromeres in a BRN1 mutant background (Ouspenski complex might contribute to ensure proper function of the et al., 2000). These results suggest that condensin is not centromere. In S. cerevisiae, BRN1 the homologue of Barren, required for the formation of functional kinetochores and that has been implicated in the formation of functional mitotic at metaphase-anaphase transition sister centromeres disjoin kinetochores (Ouspenski et al., 2000) and in C. elegans normally and segregate but sister chromatid arms remain condensin activity is required for the normal orientation of the attached causing stretching of the centromeres. centromere towards the mitotic spindle (Hagstrom et al., 2002). Immunofluorescence and biochemical studies have Here we show that depletion of DmSMC4 does not affect the suggested that condensed chromatids contain a central axial localisation of centromere or kinetochore proteins and that structure (Paulson and Laemmli, 1977). Topoisomerase II microtubules associate with kinetochores. Furthermore, we (Earnshaw et al., 1985; Gasser et al., 1986) and condensin 4774 Journal of Cell Science 116 (23) (Hirano and Mitchison, 1994; Steffensen et al., 2001) have DmSMC4 depletion (Clarke et al., 1993; Uemura et al., 1986; been identified at this elusive structure. Previously we showed Uemura et al., 1987). Accordingly, we believe that the that in Drosophila S2 cells condensins associate with chromatin bridges observed after depletion of DmSMC4 are chromatin at prophase localising to the axis of sister due to inappropriate activity of Topo II resulting in the chromatids throughout mitosis (Steffensen et al., 2001). In this maintenance of catenated DNA between sister chromatids. study we show that Topo II also localises to the axis of sister Previous reports have suggested a possible mechanistic chromatids throughout mitosis. However, in prometaphase interaction between cohesins and condensins (Castano et al., chromosomes it is clear that condensin and Topo II only show 1996; Lavoie et al., 2002). However, we show here that partial co-localisation. DmSMC4 and Topo II localise to depletion of DmSMC4 does not alter the localisation or discrete sites that alternate along the chromatid axis. Although removal of cohesins from mitotic chromatin in Drosophila S2 similar patterns of localisation has been recently described for cells. Similarly, in S. cerevisiae it has been shown that although hBarren and Topo IIα in HeLa cells, hBarren appears to bind sister chromatid separation does not occur normally in Ycs4 chromatin only during prometaphase while Topo IIα is present mutants, MCD1/SCC1 is released from chromosomes at from prophase (Maeshima and Laemmli, 2003). This the metaphase-anaphase transition (Bhalla et al., 2002). discrepancy in the kinetics of condensin accumulation at early Conversely, depletion of cohesins in higher eukaryotes does stages of mitosis probably represents cell type-specific not appear to affect chromosome condensation (Losada et al., differences since it is unlikely that SMC and non-SMC 1998; Sonoda et al., 2001; Vass et al., 2003) and in Xenopus subunits bind chromatin independently (see above). extracts the release of cohesin during prophase is not required Furthermore, we show here that depletion of DmSMC4 for chromatin compaction mediated by condensin (Losada abolishes the localisation of Topo II to a well-defined axial et al., 2002). Taken together, these results indicate that the structure even though there is no significant reduction in the removal of cohesins during mitosis is independent of level of chromatin-associated Topo II. Similarly, in yeast, condensin activity. localisation of Topo II to mitotic chromatin has been shown to Depletion of cohesins causes premature sister chromatid depend upon condensin function (Bhalla et al., 2002). separation and a significant prometaphase arrest (Sonoda et al., However, more recent data has suggested that in chromatin 2001; Vass et al., 2003). This prometaphase arrest could be due assembled in Xenopus extracts, Topo II localisation to an axial to the activity of the spindle checkpoint, which prevents exit structure of chromatids occurs independently of condensin from mitosis if proper chromosome orientation and organisation (Cuvier and Hirano, 2003). These apparently contradictory of a metaphase plate is not achieved. Strikingly, we observed results could be explained if condensin was not completely that simultaneous depletion of DmSMC4 and DRAD21 does depleted in the Xenopus extracts, allowing partial accumulation not lead to premature sister chromatid separation or arrest of Topo II to an axial chromatin structure. Since in our RNAi during prometaphase but cells progress into anaphase and depletion studies we did not find DmSMC4 either by telophase showing extensive chromatin bridges. We propose immunoflourescence or western blotting, our results suggest that sister chromatids do not separate prematurely in the that condensin plays an essential role in the organisation of the absence of cohesins because, as shown above, depletion chromatin so that Topo II can localise to the chromatid axis. of DmSMC4 prevents sister chromatid resolution by This structure is likely to be highly dynamic since recent live compromising Topo II activity. These cells then proceed into imaging and FRAP analysis in mammalian cells shows that prometaphase, kinetochores can now bind spindle microtubules Topo IIα exchanges rapidly between a cytoplasmic pool and and chromosomes congress to the metaphase plate, allowing that bound to chromosomes and centromeres (Christensen et cells to satisfy the spindle checkpoint and initiate mitotic exit. al., 2002; Tavormina et al., 2002). In vivo analysis of condensin Thus, in the absence of DmSMC4, abnormal decatenation of accumulation to the axis of sister chromatids should provide sister chromatids appears to provide an alternate mechanism to valuable insights on the dynamics of this ‘ill-defined’ structure. hold sisters together during early stages of mitosis. The abnormal distribution of Topo II to mitotic chromatin From our results we propose that condensin is essential to resulting from depletion of DmSMC4 prompted us to ask organise a clearly defined axial structure of sister chromatids whether its DNA decatenation activity was also compromised. where Topo II can localise. In the absence of this specific We showed, using an in vitro assay, that DNA decatenation localisation, Topo II can still bind chromatin but its activity of the endogenous Topo II is significantly reduced when decatenation activity is not specifically directed and sister DmSMC4 is depleted. Although these results are compatible chromatids cannot resolve properly. with a direct interaction between DmSMC4 and Topo II we were unable to detect co-immunoprecipitation (data not shown) We are grateful to Ana Tomás, for providing the Leishmania indicating that the interaction might be indirect. Nevertheless, infantum cultures and for the help in kDNA extraction, Gary Karpen our results suggest that proper activity of the enzyme requires for anti-CID antibody and Hugo Bellen for anti-Barren antibody. We condensin. A more direct interaction was reported previously thank F. Casares, C. Lopes, H. Maiato, H. Bousbaa, T. Moutinho, S. Steffensen and P. Sampaio for valuable comments on the manuscript. since it was shown that Barren interacts in a yeast two-hybrid This work was supported by grants from the Fundação para a Ciência assay with Topo II and promotes its decatenating activity (Bhat e a Tecnologia of Portugal. P. A. Coelho is supported by a grant from et al., 1996). However, it has been shown that BRN1, the yeast FCT of Portugal. homologue, is not required for Topo II activity in vivo (Lavoie et al., 2000). 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