Condensin Architecture and Interaction with DNA
2002; Elsevier BV; Volume: 12; Issue: 6 Linguagem: Inglês
10.1016/s0960-9822(02)00719-4
ISSN1879-0445
AutoresShige H. Yoshimura, Kohji Hizume, Akiko Murakami, Takashi Sutani, Kunio Takeyasu, Mitsuhiro Yanagida,
Tópico(s)RNA Research and Splicing
ResumoCondensin and cohesin are two protein complexes that act as the central mediators of chromosome condensation and sister chromatid cohesion, respectively. The basic underlying mechanism of action of these complexes remained enigmatic. Direct visualization of condensin and cohesin was expected to provide hints to their mechanisms. They are composed of heterodimers of distinct structural maintenance of chromosome (SMC) proteins and other non-SMC subunits. Here, we report the first observation of the architecture of condensin and its interaction with DNA by atomic force microscopy (AFM). The purified condensin SMC heterodimer shows a head-tail structure with a single head composed of globular domains and a tail with the coiled-coil region. Unexpectedly, the condensin non-SMC trimers associate with the head of SMC heterodimers, producing a larger head with the tail. The heteropentamer is bound to DNA in a distributive fashion, whereas condensin SMC heterodimers interact with DNA as aggregates within a large DNA-protein assembly. Thus, non-SMC trimers may regulate the ATPase activity of condensin by directly interacting with the globular domains of SMC heterodimer and alter the mode of DNA interaction. A model for the action of heteropentamer is presented. Condensin and cohesin are two protein complexes that act as the central mediators of chromosome condensation and sister chromatid cohesion, respectively. The basic underlying mechanism of action of these complexes remained enigmatic. Direct visualization of condensin and cohesin was expected to provide hints to their mechanisms. They are composed of heterodimers of distinct structural maintenance of chromosome (SMC) proteins and other non-SMC subunits. Here, we report the first observation of the architecture of condensin and its interaction with DNA by atomic force microscopy (AFM). The purified condensin SMC heterodimer shows a head-tail structure with a single head composed of globular domains and a tail with the coiled-coil region. Unexpectedly, the condensin non-SMC trimers associate with the head of SMC heterodimers, producing a larger head with the tail. The heteropentamer is bound to DNA in a distributive fashion, whereas condensin SMC heterodimers interact with DNA as aggregates within a large DNA-protein assembly. Thus, non-SMC trimers may regulate the ATPase activity of condensin by directly interacting with the globular domains of SMC heterodimer and alter the mode of DNA interaction. A model for the action of heteropentamer is presented. The SMC family of proteins plays a fundamental role in chromosome structure and dynamics [1Hirano T. SMC-mediated chromosome mechanics: a conserved scheme from bacteria to vertebrates?.Genes Dev. 1999; 13: 11-19Crossref PubMed Scopus (198) Google Scholar, 2Jessberger R. Frei C. Gasser S.M. Chromosome dynamics: the SMC protein family.Curr. Opin. Genet. Dev. 1998; 8: 254-259Crossref PubMed Scopus (79) Google Scholar, 3Strunikov A.V. SMC proteins and chromosome structure.Trends Cell Biol. 1998; 8: 454-459Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar]. They have two globular domains, each of which contains an ATP binding motif, at both termini and a middle long coiled-coil region with a central hinge [4Hopfner K.P. Karcher A. Shin D.S. Craig L. Arthur L.M. Carney J.P. Tainer J.A. Structural biology of Rad50 ATPase: ATP-driven conformational control in DNA double-strand break repair and the ABC-ATPase superfamily.Cell. 2000; 101: 789-800Abstract Full Text Full Text PDF PubMed Scopus (810) Google Scholar]. Bacterial SMC forms a homodimer [5Melby T.E. Ciampaglio C.N. Briscoe G. Erickson H.P. The symmetrical structure of structural maintenance of chromosomes (SMC) and MukB proteins: long, antiparallel coiled coils, folded at a flexible hinge.J. Cell Biol. 1998; 142: 1595-1604Crossref PubMed Scopus (332) Google Scholar, 6Hirano M. Anderson D.E. Erickson H.P. Hirano T. Bimodal activation of SMC ATPase by intra- and inter-molecular interactions.EMBO J. 2001; 20: 3238-3250Crossref PubMed Scopus (141) Google Scholar], and the coiled-coil rod has a flexible central hinge, allowing a "scissoring" action. Eukaryotic SMC may use a similar organizational principle, but its architecture has not been reported. In fission yeast, mutations in SMC proteins (Cut3 and Cut14) were found to block chromosome condensation and sister chromatid separation in mitosis [7Saka Y. Sutani T. Yamashita Y. Saitoh S. Takeuchi M. Nakaseko Y. Yanagida M. Fission yeast cut3 and cut14, members of the ubiquitous protein family, are required for chromosome condensation and segregation in mitosis.EMBO J. 1994; 13: 4938-4952Crossref PubMed Scopus (286) Google Scholar]. The purified Cut3-Cut14 heterodimer showed renaturation activity of single-stranded DNAs but no ATPase activity [8Sutani T. Yanagida M. DNA renaturation activity of the SMC complex implicated in chromosome condensation.Nature. 1997; 388: 798-801Crossref PubMed Scopus (117) Google Scholar]. The SMC proteins have been identified as components of condensin and cohesin complexes in a variety of eukaryotes [9Hirano T. Chromosome cohesion, condensation, and separation.Annu. Rev. Biochem. 2000; 69: 115-144Crossref PubMed Scopus (226) Google Scholar, 10Koshland D. Strunnikov A. Mitotic chromosome condensation.Annu. Rev. Cell Dev. Biol. 1996; 12: 305-333Crossref PubMed Scopus (286) Google Scholar, 11Nasmyth K. Separating sister chromatids.Trends Biochem. Sci. 1999; 24: 98-104Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 12Losada A. Hirano M. Hirano T. Identification of Xenopus SMC protein complexes required for sister chromatid cohesion.Genes Dev. 1998; 12: 1986-1997Crossref PubMed Scopus (519) Google Scholar, 13Tomonaga T. Nagao K. Kawasaki Y. Furuya K. Murakami A. Morishita J. Yuasa T. Sutani T. Kearsey S.E. Uhlmann F. et al.Characterization of fission yeast cohesin: essential anaphase proteolysis of Rad21 phosphorylated in the S phase.Genes Dev. 2000; 14: 2757-2770Crossref PubMed Scopus (240) Google Scholar, 14Hirano T. Kobayashi R. Hirano M. Condensins, chromosome condensation protein complexes containing XCAP-C, XCAP-E and a Xenopus homologue of the Drosophila Barren protein.Cell. 1997; 89: 511-521Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar, 15Sutani T. Yuasa T. Tomonaga T. Dohmae N. Takio K. Yanagida M. Fission yeast condensin complex: essential roles of non-SMC subunits for condensation and Cdc2 phosphorylation of Cut3/SMC4.Genes Dev. 1999; 13: 2271-2283Crossref PubMed Scopus (213) Google Scholar]. Condensin and cohesin complex contain three and two non-SMC subunits, respectively [12Losada A. Hirano M. Hirano T. Identification of Xenopus SMC protein complexes required for sister chromatid cohesion.Genes Dev. 1998; 12: 1986-1997Crossref PubMed Scopus (519) Google Scholar, 13Tomonaga T. Nagao K. Kawasaki Y. Furuya K. Murakami A. Morishita J. Yuasa T. Sutani T. Kearsey S.E. Uhlmann F. et al.Characterization of fission yeast cohesin: essential anaphase proteolysis of Rad21 phosphorylated in the S phase.Genes Dev. 2000; 14: 2757-2770Crossref PubMed Scopus (240) Google Scholar, 14Hirano T. Kobayashi R. Hirano M. Condensins, chromosome condensation protein complexes containing XCAP-C, XCAP-E and a Xenopus homologue of the Drosophila Barren protein.Cell. 1997; 89: 511-521Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar, 15Sutani T. Yuasa T. Tomonaga T. Dohmae N. Takio K. Yanagida M. Fission yeast condensin complex: essential roles of non-SMC subunits for condensation and Cdc2 phosphorylation of Cut3/SMC4.Genes Dev. 1999; 13: 2271-2283Crossref PubMed Scopus (213) Google Scholar, 16Freeman L. Aragon-Alcaide L. Strunnikov A. The condensin complex governs chromosome condensation and mitotic transmission of rDNA.J. Cell Biol. 2000; 149: 811-824Crossref PubMed Scopus (241) Google Scholar]. To purify the heterodimer, Cut3 and Cut14 were simultaneously expressed in Schizosaccharomyces pombe cells under the inducible promoter nmt1[8Sutani T. Yanagida M. DNA renaturation activity of the SMC complex implicated in chromosome condensation.Nature. 1997; 388: 798-801Crossref PubMed Scopus (117) Google Scholar]. The C terminus of Cut14 was tagged with hemagglutinin antigen (HA) and hexa-histidine (H6) for affinity purification (Experimental Procedures). The tagged molecules used were all functional, as they were able to replace the wild-type genes (data not shown). The cell extract containing Cut3-Cut14 HAH6 was loaded onto a Ni-affinity chromatography column and eluted by imidazole, followed by size fractionation. Purity of the isolated Cut3 and Cut14 complex was high, judging from staining patterns by Coomassie brilliant blue (CBB, Figure 1A). The ATPase domain is fully retained in a partially cleaved Cut3 [8Sutani T. Yanagida M. DNA renaturation activity of the SMC complex implicated in chromosome condensation.Nature. 1997; 388: 798-801Crossref PubMed Scopus (117) Google Scholar]. The observation of the purified heterodimer by AFM revealed abundant head-tail particles (Figure 1B). The head-tail structure can be classified into two major types: long tail (∼45 nm) in 70% and short tail (∼25 nm) in the remaining population (Figure 1C). The long tail could often be resolved into two, suggesting that it consisted of folded coiled-coil regions. However, the open V-shaped structure frequently seen for prokaryotic SMC homodimers [5Melby T.E. Ciampaglio C.N. Briscoe G. Erickson H.P. The symmetrical structure of structural maintenance of chromosomes (SMC) and MukB proteins: long, antiparallel coiled coils, folded at a flexible hinge.J. Cell Biol. 1998; 142: 1595-1604Crossref PubMed Scopus (332) Google Scholar] was never observed. The 45 nm tail occasionally kinked showed a protrusion on the tip, possibly representing the hinge (Figure 1B, open triangle). The average values of the tail length and the head diameter (∼28 nm) were obtained (Figure 1D). The total length of the coiled-coil regions predicted from the amino acid sequences of Cut3 and Cut14 is ∼90 nm (∼600 aa), approximately twice the ∼45 nm tail length. One model (Figure 1E) thus is that the heterodimer is folded back at the central hinge such that the single head contains four globular domains. The real diameter of images is known to be exaggerated by the "tip effect" of an AFM probe: a calculation subtracting the effect [17Bustamante C. Keller D. Yang C. Scanning force microscopy of nucleic acids and nucleoprotein assemblies.Curr. Opin. Struct. Biol. 1993; 3: 363-372Crossref Scopus (115) Google Scholar] suggests that the head corresponds to a molecular mass of ∼110 kDa, similar to the total molecular weight of the four globular domains of the Cut3/Cut14. For the ∼25 nm short tail particle, further folding of the coiled-coil region may take place, possibly due to the interaction between the hinge and the head. For comparison, the SMC heterodimer of cohesin complex (Psm1-Psm3) was overproduced and purified. The isolated cohesin SMC heterodimer Psm1-Psm3 had also a head-tail appearance (see the Supplementary Material available with this article online). As Rad21/Scc1 was highly proteolytic during the preparation, the cohesin holocomplex [13Tomonaga T. Nagao K. Kawasaki Y. Furuya K. Murakami A. Morishita J. Yuasa T. Sutani T. Kearsey S.E. Uhlmann F. et al.Characterization of fission yeast cohesin: essential anaphase proteolysis of Rad21 phosphorylated in the S phase.Genes Dev. 2000; 14: 2757-2770Crossref PubMed Scopus (240) Google Scholar] has not been observed. To purify the trimeric non-SMC complex of condensin, if it is formed, we constructed a S. pombe strain which overproduced simultaneously three subunits: Cnd1, Cnd2, and Cnd3. The amino terminus of the Cnd2 was tagged with 6H, and the tagged protein was shown to be functional (data not shown). The Ni column followed by Superose gel filtration allowed us to isolate the trimeric complex. AFM showed that the trimeric complex formed spherical particles (see Supplementary Material). Statistical analysis of the diameters produced two peaks in a histogram. A similar calculation suggests that the diameter of the 36 nm particle corresponds to a molecular mass of 350 kDa, which fits to the trimer. The 31 nm particle might have lost Cnd2. Three non-SMC subunits can polymerize into a trimer without the SMC heterodimer. The heteropentamer was also purified using the tagged Cnd2 for affinity purification. Cnd2-HAH6 was simultaneously overexpressed with the four other subunits, Cut3, Cut14, Cnd1, and Cnd3, in S. pombe cells by the inducible promoter nmt1. The isolated condensin subunits were coeluted in gel filtration. SDS-PAGE analysis and immunoblotting revealed five subunits with the expected MWs (Figure 2A). Cut14 and Cnd1 migrate together as a single thick band when it is stained by CBB. Thus, the purified heteropentamer complex contains almost equal ratios of the five subunits. The fraction with the highest purity (fraction 22) was used for AFM. The condensin complex showed basically two types of images, as shown in Figure 2B. The common type had a large head with a 45 nm or a 25 nm tail giving a "tadpole" like appearance (Figures 2C and 2D). The head (the average diameter, 36 ± 0.6 nm) is significantly larger than that of SMC heterodimer and has almost an identical size to that of the non-SMC trimer, indicating that the non-SMC trimer binds to the head of SMC heterodimer to form a heteropentamer. A "paired" structure with two heads and two tails was also found infrequently (less than 1%; examples shown in Figure 2B, bottom row). A model for these images is depicted in Figure 2E. The non-SMC trimer associates with the head or a coiled-coil region near the head [18Hopfner K.P. Karcher A. Craig L. Woo T.T. Carney J.P. Tainer J.A. Structural biochemistry and interaction architecture of the DNA double-strand break repair Mre11 nuclease and Rad50-ATPase.Cell. 2001; 105: 473-485Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar]. Similar to the SMC heterodimer, the tip of the 45 nm tail associates with the head to form a short tail particle. To study the interaction in vitro between condensin and DNA, we first performed AFM observation of the heterodimer bound to ∼1 μm long (∼3.3 kb) duplex DNA derived from the innermost centromere region of S. pombe chromosome I. Characteristically, a large assembly of heterodimers was observed in association with DNA (Figure 3A). Such an aggregate formation among heterodimers was not seen in the absence of DNA, indicating that the assembly appears to be DNA dependent. Similar protein aggregates were made with noncentromeric DNA so that the sequence specificity is not high (data not shown). Importantly, the path of DNA free from protein association was much shorter than 1 μm (346 ± 15 nm), indicating that DNA within the aggregate was folded (Figure 3B). Taken together, the mode of association between heterodimers and DNA and between heterodimers seemed to be cooperative or the all-or-none type. Many DNA molecules were either free from protein or bound into a large assembly. This might be due to enhanced protein-protein interaction between heterodimers upon interaction with DNA [6Hirano M. Anderson D.E. Erickson H.P. Hirano T. Bimodal activation of SMC ATPase by intra- and inter-molecular interactions.EMBO J. 2001; 20: 3238-3250Crossref PubMed Scopus (141) Google Scholar, 19Hirano M. Hirano T. ATP-dependent aggregation of single-stranded DNA by a bacterial SMC homodimer.EMBO J. 1998; 17: 7139-7148Crossref PubMed Scopus (106) Google Scholar]. Alternatively, a conformational change of DNA, which facilitated association of heterodimers, might take place. The gel-shift assay was done for the purified condensin SMC heterodimer as well as the heteropentamer (Figure 3C). The 32P-labeled double-strand DNA was mixed and incubated with different concentrations of SMC heterodimer (left) and heteropentamer (right) and was run in native 8% acrylamide gel electrophoresis. Autoradiography shows the highly shifted band close to the well for the SMC heterodimer (open arrow). Higher concentrations of the SMC heterodimer increased the intensity of the band and produced no intermediate band. These results are consistent with the AFM observation that large protein aggregates were seen in a subpopulation of 3.3 kb long double-strand DNA molecules (Figure 3B). In sharp contrast, the heteropentamer complex produced no band close to the well but faint intermediate bands (filled arrows), suggesting that DNA is bound to one or two complexes in a distributive fashion. AFM observation shows that the heteropentamer complex was dispersed in association with DNA (Figure 4A). Protein aggregates were not seen, and the individual tadpole-like particles bound to DNA could be observed. This is consistent with the gel-shift assay of the heteropentamer (Figure 3C). The tip of the tail seemed to be bound to DNA, indicating that the hinge and/or nearby coiled-coil region are the sites for scattered association of the heteropentamer with DNA. We presumed that non-SMC trimers played a negative regulatory role for the SMC heterodimer to form a large assembly on DNA. We then measured the ATPase activity of the heteropentamer complex purified by a Ni column followed by gel filtration, in the absence (hatched columns, Figure 4B) or the presence (open columns) of DNA (mixture of linear and circular duplex DNA). The condensin SMC heterodimer itself did not produce any ATPase activity [8Sutani T. Yanagida M. DNA renaturation activity of the SMC complex implicated in chromosome condensation.Nature. 1997; 388: 798-801Crossref PubMed Scopus (117) Google Scholar]. The fraction 22 showing the highest ATPase activity contained the most purified heteropentamer, but the fraction 25 also assayed contained the activity. Immunodepletion of the fraction 22 by anti-HA immunobeads abolished the ATPase activity. The addition of DNA stimulated the ATPase activity by 2- to 4-fold. These results indicated that, although the heteropentamer was obtained from growing cells largely consisting of interphase cells, it did have the DNA-stimulated ATPase activity as frog M phase condensin does[20Kimura K. Hirano T. ATP-dependent positive supercoiling of DNA by 13S condensin: a biochemical implication for chromosome condensation.Cell. 1997; 90: 625-634Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar], though the specific activity was roughly one-fifth of the frog M phase condensin. AFM did not produce any recognizable structural differences in the heteropentamer in the absence or the presence of ATP (data not shown). The overproduction system for mitotic heteropentamer has not been successful. This is the first analysis of condensin architecture and the interaction with DNA by the combined methods of AFM and biochemical and genetical analysis. AFM observation of condensin SMC heterodimer and heteropentamer established that the non-SMC heterotrimer subunits were bound to the globular domains of the heterodimer. Thus, the ATPase domains of the heterodimer could be structurally regulated by the non-SMC trimer. The head opening that is observed in bacterial SMC might be regulated by the non-SMC subunits in eukaryotic condensin and cohesin. In the heteropentamer, the non-SMC trimer restrains the accessibility of head domain to the DNA, which results in dispersed binding along the DNA via the hinge region (Figure 4A). Our results are consistent with association of the head of SMC-like Rad50 with Mre11 nuclease [18Hopfner K.P. Karcher A. Craig L. Woo T.T. Carney J.P. Tainer J.A. Structural biochemistry and interaction architecture of the DNA double-strand break repair Mre11 nuclease and Rad50-ATPase.Cell. 2001; 105: 473-485Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar] and requirement of non-SMC subunits for the condensin ATPase activation [21Kimura K. Hirano T. Dual roles of the 11S regulatory subcomplex in condensin functions.Proc. Natl. Acad. Sci. USA. 2000; 97: 11972-11977Crossref PubMed Scopus (121) Google Scholar]. It is tempting to speculate that the opening of the SMC heterodimer head occurs for chromosome condensation in mitosis and is regulated by a transient gate opening by the non-SMC trimer in the presence of ATP in order to enclose the duplex DNAs for forming a loop (Figure 4C). Mitotic condensation is possibly caused by the formation of many DNA loops. For the case of cohesin, the cleavage of a non-SMC protein Scc1/Rad21 might allow transient opening of SMC heterodimer for releasing the separated chromatid DNAs. AFM employed in this study was based on the conventional air-drying procedure and could be improved with better resolution by newly developing liquid methods [22Bustamante C. Smith S.B. Liphardt J. Smith D. Single-molecule studies of DNA mechanics.Curr. Opin. Struct. Biol. 2000; 10: 279-285Crossref PubMed Scopus (725) Google Scholar, 23Engel A. Gaub H.E. Muller D.J. Atomic force microscopy: a forceful way with single molecules.Curr. Biol. 1999; 9: R133-R136Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar]. Further biochemical and ultrastructural study may elucidate cooperative protein-DNA assembly induced by aggregation of condensin SMC heterodimers, which might mimic chromosome condensation. Such an assumption is not contradictory to the activity of ATP-dependent, positive DNA supercoiling of frog 13S condensin, because the supercoiling is not catalytic and requires a stoichiometric amount of condensin [20Kimura K. Hirano T. ATP-dependent positive supercoiling of DNA by 13S condensin: a biochemical implication for chromosome condensation.Cell. 1997; 90: 625-634Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 24Kimura K. Rybenkov V.V. Crisona N.J. Hirano T. Cozzarelli N.R. 13S condensin actively reconfigures DNA by introducing global positive writhe: implications for chromosome condensation.Cell. 1999; 98: 239-248Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar]. DNA-dependent aggregation of condensin SMC heterodimer resembles intermolecular interaction of bacterial SMC, which seems to activate ATPase [6Hirano M. Anderson D.E. Erickson H.P. Hirano T. Bimodal activation of SMC ATPase by intra- and inter-molecular interactions.EMBO J. 2001; 20: 3238-3250Crossref PubMed Scopus (141) Google Scholar]. Further work definitely requires the use of the heteropentamer from the M phase cells to understand how the complex is mechanistically activated from the interphase state. Simultaneous overproduction and purification of SMC subunits of condensin were performed as described previously [8Sutani T. Yanagida M. DNA renaturation activity of the SMC complex implicated in chromosome condensation.Nature. 1997; 388: 798-801Crossref PubMed Scopus (117) Google Scholar]. The non-SMC genes cnd1+, cnd2+, and cnd3+ were fused to the nmt1 promoter and tandemly inserted into pUC119 with the ura4+ marker and the ars1. The cnd2+ gene was N-terminally tagged with six-histidine sequence for affinity purification (plasmid pET129). For the production of heteropentamer complex in yeast, the cut3+ and cut14+ genes, each of which is fused to the nmt1 promoter, were tandemly inserted into the pUC119 vector with the LEU2 marker gene and the ars1 sequence (plasmid pET115). The non-SMC genes cnd1+, cnd2+, and cnd3+ were also fused to the nmt1 promoter and similarly cloned into pUC119 with the ura4+ marker and the ars1 (plasmid pET110). The cnd2+ gene was C-terminally tagged with HA and six-histidine sequences for affinity purification. The fission yeast expressing the condensin subunits was cultured in minimal EMM2 medium in the absence of thiamine for 30 hr at 26°C. Subsequent purification steps were done at 0°C–4°C. Cells (∼2 × 1010) were washed once in lysis buffer (40 mM Tris at pH 7.5, 60 mM β-glycerophosphate, 50 mM NaCl, 2 mM MgCl2, 10% Glycerol, 0.1% NP-40) and disrupted by glass beads in 12 ml of lysis buffer containing protease inhibitors (1 mM PMSF and protease inhibitor cocktail; Sigma). The cell lysates were centrifuged at 40,000 rpm for 1 hr using a Beckman 60Ti rotor, and the supernatant was bound to 1 ml of Ni-NTA agarose (Qiagen) for 2 hr. The resin was washed twice with 10 ml of the lysis buffer containing 20 mM imidazole, and the bound proteins were eluted with 5 ml of the lysis buffer containing 200 mM imidazole. A fraction containing condensin subunits was concentrated with Microcon 50 (Amicon) and injected to Superose 6 (HR 10/30; Amersham Pharmacia) equilibrated with the buffer S (20 mM Tris-Cl [pH 7.5], containing 200 mM NaCl, 10% Glycerol, 0.02% NP-40, 1 mM 2-mercaptoethanol, and 0.1 mM PMSF). Two complementary oligonucleotides (5′-32P-CCCTATAACCCCTGCATTGAATTCCAGTCTGATAA-3′ and 5′-TTATCAGACTGGAATTCAATGCAGGGGTTATAGGG-3′) were annealed in 10 mM Tris at pH 7.5 containing 50 mM NaCl, 10 mM MgCl2, and 1 mM dithiothreitol (DTT), and purified by polyacrylamide gel electrophoresis. The end-labeled double-stranded DNA was incubated with condensin protein complex purified by gel filtration in total 20 μl of the binding buffer (20 mM Tris at pH 7.5, 50 mM NaCl, 2 mM MgCl2, 10% glycerol, 1 mM DTT, 0.1 mg/ml BSA) at the room temperature for 1 hr. The sample was run in 8% nondenaturing polyacrylamide gel, followed by autoradiography. The ATPase assay was performed based on the reference [20Kimura K. Hirano T. ATP-dependent positive supercoiling of DNA by 13S condensin: a biochemical implication for chromosome condensation.Cell. 1997; 90: 625-634Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar]. Purified condensin complex was diluted with 5 mM HEPES (pH 7.4) and incubated on ice for 30 min with or without the indicated amount of DNA (3.3 kb PCR fragment containing a centromere region of S. pombe chromosome I). Fixation with 0.25%–0.5% glutaraldehyde for 1–7 hr on ice was done when necessary. For the AFM imaging, 0.5–1 ng of condensin complex was applied on the freshly cleaved mica surface, which is pretreated with 10 mM spermidine. After 10 min, the mica was gently washed with water and dried with nitrogen gas. The AFM imaging was performed with Nanoscope IIIa (Digital Instrument, CA) with a type E scanner under the Tapping ModeTM in air at room temperature. The AFM probes made of single silicon crystal with a cantilever length of 129 μm and a spring constant 33–62 N/m (OLYMPUS) were used. Images were collected in height mode and stored in the 512 × 512 pixel format. The images obtained were then plane fitted and analyzed by the computer program accompanied with the imaging module. Supplementary Material including figures showing purification and AFM observation of SMC heterodimer of cohesin and purification and AFM observation of the non-SMC heterotrimer complex can be found online at http://images.cellpress.com/supmat/supmatin.htm. This study was supported by the CREST research project of the Japan Science and Technology Corporation (to M.Y. and to K.T), the Special Co-ordination Funds (to K.T), and the COE Research Grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan. S.H.Y and T.S. are recipients of the Japan Society for the Promotion of Science pre- and postdoctoral fellowships, respectively. 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