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Chromosome Condensation by a Human Condensin Complex inXenopus Egg Extracts

2001; Elsevier BV; Volume: 276; Issue: 8 Linguagem: Inglês

10.1074/jbc.c000873200

1083-351X

Keiji Kimura, Olivier Cuvier, Tatsuya Hirano,

DNA Repair Mechanisms

13S condensin is a five-subunit protein complex that plays a central role in mitotic chromosome condensation. The condensin complex was originally identified and purified fromXenopus egg extracts and shown to have an ATP-dependent positive supercoiling activity in vitro. We report here the characterization of a human condensin complex purified from HeLa cell nuclear extracts. The human 13S complex has exactly the same composition as its Xenopuscounterpart, being composed of two structural maintenance of chromosomes (human chromosome-associated polypeptide (hCAP)-C and hCAP-E) subunits and three non-structural maintenance of chromosomes (hCAP-D2/CNAP1, hCAP-G, and hCAP-H/BRRN) subunits. Human condensin purified from asynchronous HeLa cell cultures fails to reconfigure DNA structure in vitro. When phosphorylated by purified cdc2-cyclin B, however, it gains the ability to introduce positive supercoils into DNA in the presence of ATP and topoisomerase I. Strikingly, human condensin can induce chromosome condensation when added back into a Xenopus egg extract that has been immunodepleted of endogenous condensin. Thus, the structure and function of the condensin complex are highly conserved betweenXenopus and humans, underscoring its fundamental importance in mitotic chromosome dynamics in eukaryotic cells.AF331796 13S condensin is a five-subunit protein complex that plays a central role in mitotic chromosome condensation. The condensin complex was originally identified and purified fromXenopus egg extracts and shown to have an ATP-dependent positive supercoiling activity in vitro. We report here the characterization of a human condensin complex purified from HeLa cell nuclear extracts. The human 13S complex has exactly the same composition as its Xenopuscounterpart, being composed of two structural maintenance of chromosomes (human chromosome-associated polypeptide (hCAP)-C and hCAP-E) subunits and three non-structural maintenance of chromosomes (hCAP-D2/CNAP1, hCAP-G, and hCAP-H/BRRN) subunits. Human condensin purified from asynchronous HeLa cell cultures fails to reconfigure DNA structure in vitro. When phosphorylated by purified cdc2-cyclin B, however, it gains the ability to introduce positive supercoils into DNA in the presence of ATP and topoisomerase I. Strikingly, human condensin can induce chromosome condensation when added back into a Xenopus egg extract that has been immunodepleted of endogenous condensin. Thus, the structure and function of the condensin complex are highly conserved betweenXenopus and humans, underscoring its fundamental importance in mitotic chromosome dynamics in eukaryotic cells. AF331796 8S core subcomplex structural maintenance of chromosomes chromosome-associated polypeptides 11S regulatory subcomplex expressed sequence tag polyacrylamide gel electrophoresis dithiothreitol human Xenopus Chromosome condensation is an essential cellular process that ensures the faithful segregation of chromosomes in both mitosis and meiosis (1Koshland D. Strunnikov A. Annu. Rev. Cell Dev. Biol. 1996; 12: 305-333Crossref PubMed Scopus (286) Google Scholar, 2Hirano T. Annu. Rev. Biochem. 2000; 69: 115-144Crossref PubMed Scopus (226) Google Scholar). Recent studies in Xenopus egg cell-free extracts led to the identification of a five-subunit protein complex, termed 13S condensin, that plays a key role in this process (3Hirano T. Mitchison T.J. Cell. 1994; 79: 449-458Abstract Full Text PDF PubMed Scopus (432) Google Scholar, 4Hirano T. Kobayashi R. Hirano M. Cell. 1997; 89: 511-521Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar). TheXenopus 13S condensin complex is composed of two subcomplexes, an 8S core subcomplex (8SC)1 consisting of two structural maintenance of chromosomes (SMC) subunits (XCAP-C and -E) and an 11S regulatory subcomplex (11SR) containing three non-SMC subunits (XCAP-D2, -G, and -H) (3Hirano T. Mitchison T.J. Cell. 1994; 79: 449-458Abstract Full Text PDF PubMed Scopus (432) Google Scholar, 4Hirano T. Kobayashi R. Hirano M. Cell. 1997; 89: 511-521Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar, 5Kimura K. Hirano M. Kobayashi R. Hirano T. Science. 1998; 282: 487-490Crossref PubMed Scopus (250) Google Scholar, 6Kimura K. Hirano T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11972-11977Crossref PubMed Scopus (122) Google Scholar). Similar five-subunit complexes have also been identified from Schizosaccharomyces pombe (7Sutani T. Yuasa T. Tomonaga T. Dohmae N. Takio K. Yanagida M. Genes Dev. 1999; 13: 2271-2283Crossref PubMed Scopus (213) Google Scholar) and Saccharomyces cerevisiae (8Freeman L. Aragon-Alcaide L. Strunnikov A.V. J. Cell Biol. 2000; 149: 811-824Crossref PubMed Scopus (241) Google Scholar). Each of the condensin subunits is essential for cell viability in yeasts, and their mutations lead to defects in chromosome condensation and segregation in mitosis (7Sutani T. Yuasa T. Tomonaga T. Dohmae N. Takio K. Yanagida M. Genes Dev. 1999; 13: 2271-2283Crossref PubMed Scopus (213) Google Scholar, 8Freeman L. Aragon-Alcaide L. Strunnikov A.V. J. Cell Biol. 2000; 149: 811-824Crossref PubMed Scopus (241) Google Scholar, 9Saka Y. Sutani T. Yamashita Y. Saitoh S. Takeuchi M. Nakaseko Y. Yanagida M. EMBO J. 1994; 13: 4938-4952Crossref PubMed Scopus (286) Google Scholar, 10Strunnikov A.V. Hogan E. Koshland D. Genes Dev. 1995; 9: 587-599Crossref PubMed Scopus (295) Google Scholar, 11Lavoie B.D. Tuffo K.M. Oh S. Koshland D. Holm C. Mol. Biol. Cell. 2000; 11: 1293-1304Crossref PubMed Scopus (116) Google Scholar, 12Ouspenski I.I. Cabello O.A. Brinkley B.R. Mol. Biol. Cell. 2000; 11: 1305-1313Crossref PubMed Scopus (91) Google Scholar). Subunit composition of the putative condensin complex in human cells is not fully understood, although a complex containing hCAP-C, hCAP-E, and CNAP1 (homologous to XCAP-C, XCAP-E, and XCAP-D2, respectively) has been reported very recently (13Schmiesing J.A. Gregson H.C. Zhou S. Yokomori K. Mol. Cell. Biol. 2000; 20: 6996-7006Crossref PubMed Scopus (101) Google Scholar). 13S condensin, when purified from Xenopus egg mitotic extracts, displays a DNA-stimulated ATPase activity and changes DNA structure in an ATP-dependent manner in vitro. It introduces positive supercoils into relaxed circular DNA in the presence of type I topoisomerases (14Kimura K. Hirano T. Cell. 1997; 90: 625-634Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar) and converts nicked circular DNA into positively knotted forms in the presence of a type II topoisomerase (15Kimura K. Rybenkov V.V. Crisona N.J. Hirano T. Cozzarelli N.R. Cell. 1999; 98: 239-248Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). The interphase form of 13S condensin lacks these activities, although its subunit composition is the same as that of the mitotic form. It was found that mitosis-specific phosphorylation of the non-SMC subunits by purified cdc2-cyclin B can activate the ATP-dependent activities of 13S condensin in vitro (5Kimura K. Hirano M. Kobayashi R. Hirano T. Science. 1998; 282: 487-490Crossref PubMed Scopus (250) Google Scholar, 15Kimura K. Rybenkov V.V. Crisona N.J. Hirano T. Cozzarelli N.R. Cell. 1999; 98: 239-248Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Moreover, the ability of 13S condensin to induce DNA supercoiling in the purified system is tightly coupled with its ability to promote chromosome condensation in the cell-free extracts (6Kimura K. Hirano T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11972-11977Crossref PubMed Scopus (122) Google Scholar). These results suggest strongly that the supercoiling and knotting activities are fundamental to condensin function and directly contribute to mitotic chromosome condensation. However, these in vitro activities have so far been detected only in theXenopus condensin complex purified from egg extracts. It is therefore very important to determine whether the functional, as well as structural, properties of the condensin complex are conserved in different organisms and at different developmental stages. In this paper, we report the purification of 13S condensin from HeLa cell nuclear extracts and describe its complete subunit composition. We show that the human complex displays ATP-dependent supercoiling and knotting activities that are regulated by phosphorylation by cdc2-cyclin B in vitro. Finally, a functional complementation assay demonstrates that the human condensin complex can induce chromosome condensation in Xenopus egg extracts. By searching the human expressed sequence tag (EST) data base, we identified a set of partial cDNA sequences that potentially encode the human ortholog of XCAP-G (AW503468, AW194979, AW401913, BE278549, AI628901, and AI761782). A nucleotide sequence assembled from these clones encoded a 768-amino acid polypeptide that is homologous to the C-terminal 3/4 of XCAP-G. The following two polymerase chain reaction primers were designed to amplify a human cDNA fragment using a λgt10 library as a template: 5hG1, 5′-CCCTCTAGAGCTATGCAGAAGCATCTTC-3′ (XbaI tag sequence is underlined); and 3hG1, 5′-TAGGATCCAGGGATATTGGGATTGTGGG-3′ (BamHI tag sequence is underlined). A resulting ∼530-base pair fragment was used as a hybridization probe to screen a HeLa cell cDNA library (Stratagene). Eight positive clones were analyzed, and seven of them were found to contain the full coding sequence. One of the full-length clones (pHG104) was fully sequenced. Rabbit polyclonal antisera were raised against synthetic peptides corresponding to the C-terminal sequences of hCAP-C (VAVNPKEIASKGLC; see Ref. 16Schmiesing J.A. Ball A.R. Gregson H.C. Alderton J.M. Zhou S. Yokomori K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12906-12911Crossref PubMed Scopus (93) Google Scholar), hCAP-E (KSKAKPPKGAHVEV; see Ref.16Schmiesing J.A. Ball A.R. Gregson H.C. Alderton J.M. Zhou S. Yokomori K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12906-12911Crossref PubMed Scopus (93) Google Scholar), hCAP-D2/CNAP1 (TTPILRASARRHRS; see Refs. 5Kimura K. Hirano M. Kobayashi R. Hirano T. Science. 1998; 282: 487-490Crossref PubMed Scopus (250) Google Scholar and 13Schmiesing J.A. Gregson H.C. Zhou S. Yokomori K. Mol. Cell. Biol. 2000; 20: 6996-7006Crossref PubMed Scopus (101) Google Scholar), hCAP-G (EKSKLNLAQFLNEDLS; this study), and hCAP-H/BRRN (GTEDLSDVLVRQGD; see Refs. 4Hirano T. Kobayashi R. Hirano M. Cell. 1997; 89: 511-521Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar and 17Cabello O.A. Baldini A. Bhat M. Bellen H. Belmont J.W. Genomics. 1997; 46: 311-313Crossref PubMed Scopus (9) Google Scholar). Immunization and affinity-purification of antibodies were performed as described previously (4Hirano T. Kobayashi R. Hirano M. Cell. 1997; 89: 511-521Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar). HeLa cell nuclear extracts were prepared as described previously (18Losada A. Yokochi T. Kobayashi R. Hirano T. J. Cell Biol. 2000; 150: 405-416Crossref PubMed Scopus (261) Google Scholar) in a buffer containing 20 mm K-Hepes (pH 8.0), 100 mm KCl, 2 mm MgCl2, 0.2 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride, 1 mmβ-mercaptoethanol, and 10% glycerol. After immunoaffinity purification of cohesin (18Losada A. Yokochi T. Kobayashi R. Hirano T. J. Cell Biol. 2000; 150: 405-416Crossref PubMed Scopus (261) Google Scholar), 2 ml of the flowthrough fraction (equivalent to 9 × 108 cells) were incubated with 200 μg of anti-hCAP-G coupled to 200 μl of protein A-agarose beads (Life Technologies, Inc.) at 4 °C for 1 h. The mixture was poured into a 2-ml column, washed consecutively with 80 column volumes of XBE2-gly (10 mm K-Hepes (pH 7.7), 100 mmKCl, 2 mm MgCl2, 0.1 mmCaCl2, 5 mm EGTA, and 10% glycerol), 10 volumes of XBE2-gly containing 400 mm KCl, and 10 volumes of XBE2-gly. To elute condensin from the column, the hCAP-G tail peptide was added at a final concentration of 0.4 mg/ml in XBE2-gly. The peak fractions were pooled (∼150 μl), supplemented with 0.1 mg/ml ovalbumin and 1 mm DTT, and concentrated 3-fold with Microcon-30 tubes (Amicon). This procedure yielded ∼1.5–2.0 μg of human condensin of >95% purity as judged by SDS polyacrylamide gel electrophoresis (PAGE). For phosphorylation, purified condensin (1 μg) was incubated at 22 °C for 30 min in 40 μl of phosphorylation buffer (XBE2-gly containing 1 mg/ml ovalbumin, 1 mm DTT, and 1 mm MgATP) in the presence or absence of cdc2-cyclin B (1 ng) purified from Xenopus egg extracts (5Kimura K. Hirano M. Kobayashi R. Hirano T. Science. 1998; 282: 487-490Crossref PubMed Scopus (250) Google Scholar). [γ-32P]ATP (specific radioactivity of >5000 Ci/mmol) was added at a final concentration of 0.05 mCi/ml in the labeling reaction. For the rescue experiment (see Fig. 4), condensin was eluted in XBE6 (10 mm K-Hepes (pH 7.7), 100 mm KCl, 6 mm MgCl2, 0.1 mm CaCl2, 5 mm EGTA, and 50 mm sucrose) instead of XBE2-gly and concentrated in the same way. The amounts of purified complexes were determined by SDS-PAGE followed by Coomassie Blue stain using bovine serum albumin as a standard. Immunodepletion of condensin from Xenopus egg extracts was performed as described previously (4Hirano T. Kobayashi R. Hirano M. Cell. 1997; 89: 511-521Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar, 6Kimura K. Hirano T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11972-11977Crossref PubMed Scopus (122) Google Scholar). For the rescue experiment, an amount ofXenopus or human condensin equivalent to the endogenous level was added back into the depleted extract. Sucrose gradient centrifugation, supercoiling, and knotting assays were performed as described previously (4Hirano T. Kobayashi R. Hirano M. Cell. 1997; 89: 511-521Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar, 14Kimura K. Hirano T. Cell. 1997; 90: 625-634Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar,15Kimura K. Rybenkov V.V. Crisona N.J. Hirano T. Cozzarelli N.R. Cell. 1999; 98: 239-248Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). A search of the human EST data base identified a set of partial cDNA sequences that potentially encode the human ortholog of XCAP-G, a 130-kDa subunit of theXenopus 13S condensin complex (4Hirano T. Kobayashi R. Hirano M. Cell. 1997; 89: 511-521Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar). On the basis of this information, we designed polymerase chain reaction primers, amplified a human cDNA fragment, and used it as a hybridization probe to screen a HeLa cell cDNA library. The longest open reading frame deduced from multiple clones encoded a 1,015-amino acid polypeptide with a calculated molecular mass of 114.1 kDa, which was highly homologous to XCAP-G along its entire length (62% identical; 74% conserved). We named this polypeptide human chromosome-associated polypeptide-G (hCAP-G). Members of this class of condensin subunits had been reported from S. pombe (Cnd3; see Ref. 7Sutani T. Yuasa T. Tomonaga T. Dohmae N. Takio K. Yanagida M. Genes Dev. 1999; 13: 2271-2283Crossref PubMed Scopus (213) Google Scholar) and S. cerevisiae (Ycg1/Ycs5; see Refs. 8Freeman L. Aragon-Alcaide L. Strunnikov A.V. J. Cell Biol. 2000; 149: 811-824Crossref PubMed Scopus (241) Google Scholar and 12Ouspenski I.I. Cabello O.A. Brinkley B.R. Mol. Biol. Cell. 2000; 11: 1305-1313Crossref PubMed Scopus (91) Google Scholar). An alignment of these sequences is shown in Fig. 1. We found that hCAP-G contains HEAT repeats, a highly degenerate repeating motif found in a number of proteins with diverse functions (19Andrade M.A. Bork P. Nat. Genet. 1995; 11: 115-116Crossref PubMed Scopus (467) Google Scholar) (Fig. 1,red rectangles). This is in agreement with our recent sequence analysis showing that each of the CAP-G family members has at least nine copies of this motif (20Neuwald A.F. Hirano T. Genome Res. 2000; 10: 1445-1452Crossref PubMed Scopus (231) Google Scholar). During the preparation of this manuscript, the same human cDNA was cloned by serological screening in an attempt to identify melanoma antigens (21Jager D. Stockert E. Jager E. Gure A.O. Scanlan M.J. Knuth A. Old L.J. Chen Y.-T. Cancer Res. 2000; 60: 3584-3591PubMed Google Scholar). Very recently, Schmiesing et al. (13Schmiesing J.A. Gregson H.C. Zhou S. Yokomori K. Mol. Cell. Biol. 2000; 20: 6996-7006Crossref PubMed Scopus (101) Google Scholar) reported a human protein complex that contains hCAP-C, hCAP-E, and CNAP1 (homologous to XCAP-D2; see Ref. 5Kimura K. Hirano M. Kobayashi R. Hirano T. Science. 1998; 282: 487-490Crossref PubMed Scopus (250) Google Scholar). It remains unknown, however, whether the complex also contains hCAP-G (this study) and BRNN (17Cabello O.A. Baldini A. Bhat M. Bellen H. Belmont J.W. Genomics. 1997; 46: 311-313Crossref PubMed Scopus (9) Google Scholar) (homologous to XCAP-H; see Ref. 4Hirano T. Kobayashi R. Hirano M. Cell. 1997; 89: 511-521Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar). In this manuscript, we refer to CNAP1 and BRNN as hCAP-D2 and hCAP-H, respectively, in accordance with the nomenclature of theirXenopus orthologs. We raised a set of peptide antibodies against the putative subunits of human condensin (see "Experimental Procedures"). It was found that an hCAP-G antibody coimmunoprecipitates five discrete bands from a HeLa cell nuclear extract as judged by silver stain (Fig.2 A, lane 1). Immunoblotting analysis clearly showed that the five bands correspond to hCAP-C (170 kDa), -D2 (155 kDa), -E (135 kDa), -G (120 kDa), and -H (100–105 kDa) (Fig. 2 A, lanes 2–6). The stoichiometry of the five subunits was apparently ∼1:1:1:1:1, although hCAP-H always appeared as a fuzzy band (presumably because of multiple phosphorylation) as we had also observed for XCAP-H inXenopus egg extracts (4Hirano T. Kobayashi R. Hirano M. Cell. 1997; 89: 511-521Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar). When a HeLa nuclear extract was subject to sucrose gradient centrifugation, the five polypeptides cofractionated with a sedimentation coefficient of ∼13 S (Fig. 2 B, upper panel). A small fraction of hCAP-C and hCAP-E cosedimented at a second peak of ∼8 S, suggesting the presence of an SMC core subcomplex (8SC). These sedimentation properties of the human condensin subunits were very similar to those found in Xenopus egg extracts (4Hirano T. Kobayashi R. Hirano M. Cell. 1997; 89: 511-521Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar). In this experiment, we did not detect the presence of an 11SR consisting of the non-SMC subunits only (6Kimura K. Hirano T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11972-11977Crossref PubMed Scopus (122) Google Scholar). This was not surprising, however, because even in the Xenopus egg extracts this subcomplex is present at a very low level (∼1/10 of the 13S complex) and not detectable in the same assay (4Hirano T. Kobayashi R. Hirano M. Cell. 1997; 89: 511-521Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar). We then purified the human 13S complex by immunoaffinity column chromatography using the hCAP-G peptide antibody and fractionated it by sucrose gradient centrifugation (Fig. 2 B, lower panel). Again, all the five subunits cofractionated at a single peak of 13S, confirming that they tightly associate with each other and form a complex. Taking these results together, we conclude that the 13S holocomplex of human condensin has exactly the same size and subunit composition as itsXenopus counterpart. The Xenopus 13S condensin complex introduces positive supercoils into relaxed circular DNA in the presence of ATP and topoisomerase I in vitro (supercoiling assay; see Ref. 14Kimura K. Hirano T. Cell. 1997; 90: 625-634Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). It also converts nicked circular DNA into positively knotted forms in the presence of ATP and topoisomerase II (knotting assay; see Ref. 15Kimura K. Rybenkov V.V. Crisona N.J. Hirano T. Cozzarelli N.R. Cell. 1999; 98: 239-248Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). The two activities are regulated by mitosis-specific phosphorylation of the non-SMC subunits (5Kimura K. Hirano M. Kobayashi R. Hirano T. Science. 1998; 282: 487-490Crossref PubMed Scopus (250) Google Scholar, 15Kimura K. Rybenkov V.V. Crisona N.J. Hirano T. Cozzarelli N.R. Cell. 1999; 98: 239-248Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). We wished to test whether human condensin displays a similar set of activities. When the complex was affinity-purified from a nuclear extract of an asynchronously grown HeLa cell culture (Fig.3 A, lane 1), it exhibited little activity in the supercoiling and knotting assays (Fig.3 B, lanes 1–4). We reasoned that most of the purified complexes were in the interphase (unphosphorylated) form, thereby producing the negative result. To test this possibility, the purified condensin fraction was treated with cdc2-cyclin B (Fig.3 A, lane 2). This treatment phosphorylated the three non-SMC subunits, hCAP-D2, hCAP-G, and hCAP-H, as judged by [32P] labeling (Fig. 3 A, lane 4). Remarkably, we found that the phosphorylated form of condensin was active in both of supercoiling and knotting assays (Fig. 3 B,lanes 5–8). Neither of these activities was found in the purified cdc2-cyclin B fraction alone. The apparently less effective stimulation of the knotting activity compared with the supercoiling activity (Fig. 3 B, lanes 4 and 8) is probably because of the less quantitative nature of the former assay; a similar observation was made with Xenopus condensin (15Kimura K. Rybenkov V.V. Crisona N.J. Hirano T. Cozzarelli N.R. Cell. 1999; 98: 239-248Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). As expected, two-dimensional gel electrophoresis demonstrated that the final products of the supercoiling assay were positively supercoiled (data not shown). These results show that the human condensin complex displays the same set of biochemical activities as itsXenopus counterpart. The cdc2-mediated stimulation of these activities also suggests that they contribute directly to mitosis-specific condensation of chromosomes in human somatic cells. To further test for the functional similarity between the human and Xenopus condensin complexes, we set up a complementation assay in Xenopus egg extracts. When sperm chromatin was incubated in a control extract containing endogenous condensin (Fig. 4 A, lane 1), it was converted into a mass of mitotic chromosomes (Fig.4 B, panel a). In a condensin-depleted extract (Fig. 4 A, lane 2), however, no chromosome assembly occurred (Fig. 4 B, panel b). When purified Xenopus condensin (Fig. 4 A, lane 3) was added back into the extract, it restored the ability of the extract to condense chromosomes (Fig. 4 B, panel c) as we reported previously (4Hirano T. Kobayashi R. Hirano M. Cell. 1997; 89: 511-521Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar, 6Kimura K. Hirano T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11972-11977Crossref PubMed Scopus (122) Google Scholar). Strikingly, we found that human condensin purified from a HeLa nuclear extract (Fig.4 A, lane 4) could also functionally complement the extract, inducing chromosome condensation very effectively (Fig.4 B, panel d). No pre-treatment with cdc2-cyclin B was required in this assay, suggesting that the human complex was phosphorylated by a protein kinase(s) present in the Xenopusegg extract and converted into an active complex. In summary, the current work identifies, for the first time, all the five subunits of the 13S condensin complex purified from HeLa cells. Like Xenopus condensin, the human complex displays ATP- and phosphorylation-dependent supercoiling and knotting activities in vitro, providing strong lines of evidence that they are fundamental to condensin function (and thereby mitotic chromosome condensation), not only in early embryonic cells but also in somatic cells. We thank Ana Losada for donation of HeLa cell nuclear extracts and David MacCallum and Michiko Hirano for technical assistance and instructions. We are also grateful to the members of the Hirano laboratory for critically reading the manuscript.