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Address correspondence to Tatsuya Hirano, Cold Spring Harbor Laboratory, One Bungtown Rd., Cold Spring Harbor, NY 11724. Tel.: (516) 367-8370. Fax: (516) 367-8815. E-mail: hirano{at}cshl.org
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Abstract |
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Key Words: ABC ATPases; chromosome condensation; coiled-coil; sister chromatid cohesion; structural maintenance of chromosomes
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Introduction |
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The condensin complex purified from Xenopus egg extracts binds directly to double-stranded DNA and displays a DNA-stimulated ATPase activity. Condensin has the ability to reconfigure DNA structure in an ATP-hydrolysisdependent manner. It introduces positive supercoils into relaxed circular DNA in the presence of type I topoisomerases (Kimura and Hirano, 1997), and converts nicked circular DNA into positively knotted forms in the presence of a type II topoisomerase (Kimura et al., 1999). The same set of activities has been found in the human condensin complex purified from a HeLa cell nuclear extract (Kimura et al., 2001). On the other hand, the cohesin complex displays DNA-binding properties that are remarkably different from those of condensin (Losada and Hirano, 2001). It induces the formation of large proteinDNA aggregates and stimulates intermolecular catenation (rather than knotting) of circular DNA molecules in the presence of topoisomerase II. These results are consistent with our proposal that condensin acts as an intramolecular DNA cross-linker to compact DNA, whereas cohesin acts as an intermolecular DNA cross-linker to hold two sister chromatids together (Hirano, 1999). It remains unknown, however, how the two SMC protein complexes might be able to distinguish between the two different modes of DNA interactions.
SMC proteins are conserved not only in Eukarya but also in Bacteria and Archaea (for review see Cobbe and Heck, 2000). As judged by EM, the SMC homodimer from Bacillus subtilis (BsSMC) is composed of two antiparallel coiled-coil arms connected by a flexible hinge (Melby et al., 1998). A recent biochemical analysis of BsSMC has shown that hinge-mediated opening and closing of SMC dimers may be mechanistically coupled with their dynamic interactions with DNA (Hirano et al., 2001). It is a reasonable speculation that eukaryotic SMC complexes share these structural features, but the structures have not been directly visualized until now.
In the present study, we have visualized vertebrate condensin and cohesin complexes by EM. We find that both SMC protein complexes share the two-armed structure, but display different arm conformations with characteristic hinge angles. The hinge of condensin is largely closed, whereas that of cohesin is wide open. In both complexes, the non-SMC subunits associate with the catalytic end domain(s) of the SMC dimer and not with the hinge domain. Our results suggest that condensin and cohesin have evolved to acquire differentiated structures so that they can efficiently support their specialized functions in condensation and cohesion, respectively.
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Results and discussion |
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EM of human cohesin
An antibody specific to the SMC3 subunit was used to purify the human cohesin complex from a HeLa cell nuclear extract by immunoaffinity column chromatography (Losada and Hirano, 2001). This procedure yields a large population of holocomplexes and a much smaller population of SMC1-SMC3 heterodimers, as described previously (Losada et al., 2000). An example field of the corresponding rotary shadowed molecules is shown in Fig. 2 A. Similar to condensin, cohesin had the characteristic two-armed structure of the SMC dimer. A globular complex of non-SMC subunits was associated with the SMC catalytic domains in 59 of the 66 molecules (89%) examined (Fig. 2 B). The remaining 7 molecules were interpreted to be SMC1-SMC3 heterodimers (Fig. 2 C). In striking contrast to condensin, the hinge of the cohesin complex was almost always open, and the coiled-coil arms were splayed apart. The hinge angle of the cohesin holocomplexes was measured to be 88 ± 36 (n = 52) degrees, whereas that of the condensin complexes was 6 ± 12 (n = 57) degrees. The larger standard deviation for the hinge angle of cohesin may indicate its greater flexibility compared with the hinge of condensin.
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There were two other structural differences between condensin and cohesin. First, the globular complex of non-SMC subunits was noticeably smaller for cohesin. This is consistent with the difference in the total molecular mass of the non-SMC subunits between the two complexes (350 kD for condensin and
210 kD for cohesin). Second, one arm of cohesin frequently showed a sharp bend or kink
1/3 of the way from the hinge to the catalytic domain (Fig. 2 B, arrows). The angle at the kink was measured to be 102 ± 26 (n = 52) degrees. Since a similar kink was also observed in one of the arms of the SMC1-SMC3 heterodimers (Fig. 2 C, arrows), this structural feature appears to be inherent to the SMC coiled coil.
EM of Xenopus condensin and cohesin
To confirm that the differences observed between condensin and cohesin are indeed intrinsic to the two SMC complexes, they were purified from a different source, Xenopus egg interphase extracts, and visualized by rotary shadowing. We found that the basic characters of the two Xenopus complexes were the same as those of their human counterparts. The coiled-coil arms were largely closed in the condensin holocomplexes (Fig. 3 A, first row) as well as in the SMC2-SMC4 heterodimers (Fig. 3 A, second row). In contrast, the SMC arms adopted wide-open conformations in the cohesin complexes (Fig. 3 B, first row) and in the SMC1-SMC3 heterodimers (Fig. 3 B, second row). A kink was again observed in one of the coiled-coil arms of cohesin (Fig. 3 B, arrows). Dimensions of the Xenopus holocomplexes and heterodimers were the same as their human counterparts, within 12 nm (unpublished data).
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Comparison of two-armed ATP-binding cassette (ABC) ATPases from bacteria to humans
The central hinge of the BsSMC is highly flexible, displaying a wide range of conformations from folded-rod to open-V (Melby et al., 1998; Hirano et al., 2001). In contrast, we show here that each of the two eukaryotic SMC protein complexes has its unique hinge angle: the coiled-coil arms of condensin are largely closed, whereas those of cohesin are open with an average angle of 90 degrees (Fig. 4 A). Similar, if not identical, hinge angles are observed in the corresponding SMC heterodimers, suggesting that the conformational difference between condensin and cohesin is, at least in part, intrinsic to their SMC subunits. The closed and open conformations may be determined by either the hinge structure itself, the interactions between the two coiled-coil arms, or both. In the case of condensin, for example, the closed hinge angle could be further stabilized by association of the coiled-coil arms. A sharp kink observed in one of the cohesin arms is likely to be another intrinsic property of its SMC subunits.
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The structure of the SMC protein complexes shares a number of common features with that of Rad50Mre11, a protein complex involved in double-strand DNA break repair (for review see Haber, 1998). Like SMCs, Rad50 forms a dimer in which two coiled-coil arms are connected by a central hinge, and its catalytic termini are composed of ABC ATPase domains (Hopfner et al., 2000; Lowe et al., 2001). The overall conformation of the Rad50Mre11 complex is more similar to that of cohesin than to that of condensin (Anderson et al., 2001; Hopfner et al., 2001). The hinge of the Rad50 homodimer tends to be open and the Mre11 subunits bridge the two catalytic end domains of Rad50. It is tempting to speculate that the structural similarities between Rad50Mre11 and cohesin could reflect a common mechanism of action, as both complexes have been implicated in double-strand break repair (Birkenbihl and Subramani, 1992; Sjogren and Nasmyth, 2001).
Functional implications for the actions of condensin and cohesin
On the basis of their cellular functions, we previously hypothesized that condensin and cohesin might function as intra- and intermolecular DNA cross-linkers, respectively (Hirano, 1999). DNA-binding properties of purified condensin and cohesin in vitro support this model (Kimura and Hirano, 1997; Kimura et al., 1999, 2001; Losada and Hirano, 2001). A recent mechanistic study using a bacterial SMC homodimer has shown that the two different modes of SMCDNA interactions may be coupled with global conformational changes of the SMC dimers (Hirano et al., 2001). The current EM images of "closed" condensin and "open" cohesin are consistent with this idea, and further suggest that the conformations of the two eukaryotic SMC complexes are structurally differentiated to support their specialized functions. In the case of condensin, the two catalytic domains of the closed SMC dimer may bind to contiguous DNA segments, and thereby the complex would preferentially act as an intra-molecular DNA cross-linker (Fig. 4 B, left). In the case of cohesin, an open hinge would favor the interaction of the two catalytic ends of the SMC dimer with noncontiguous DNA segments, allowing the complex to act as an intermolecular DNA cross-linker. However, almost all the images of cohesin holocomplexes show that both catalytic domains bind the non-SMC subunits, placing them in close proximity. One possibility is that, upon binding to DNA (or with a certain signal), one catalytic domain of cohesin is released from the non-SMC subunits and the wide hinge angle allows the complex to adapt a more open configuration. This conformational change would in turn facilitate the freed end to bind a DNA segment from the sister chromatid (Fig. 4 B, right, top). Moreover, as implied from the analysis of the bacterial SMC protein (Hirano et al., 2001), two SMC dimers may associate with each other, and thereby establish and strengthen the linkage between the sister chromatids (Fig. 4 B, right, bottom). In this scenario, cleavage of one of the non-SMC subunits in anaphase might destabilize the DNA binding of one or both end(s) of the cohesin complex, allowing the sister chromatids to be pulled apart by the action of the spindle microtubules. Clearly, future studies are required to determine the conformations of condensin and cohesin bound to DNA, and to understand how the DNA-binding properties are regulated by the ATP-binding and hydrolysis cycle.
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Materials and methods |
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Rotary shadowing and EM
Rotary shadowing and EM were performed as described previously (Melby et al., 1998; Anderson et al., 2001) with minor modifications. Glycerol gradient centrifugation was omitted, and fractions containing condensin or cohesin in the elution buffer were directly sprayed onto mica after adding glycerol to 40%. Specimens were rotary shadowed (Fowler and Erickson, 1979), viewed under a transmission electron microscope (Model 301; Philips Electron Optics) and photographed at 50,000x. Negatives were scanned and imported into the programs Adobe Photoshop 5.5 and NIH image for analysis and measurements of the molecules.
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Footnotes |
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Harold P. Erickson and Tatsuya Hirano contributed equally to this paper.
* Abbreviations used in this paper: ABC, ATP-binding cassette; SMC, structural maintenance of chromosomes.
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Acknowledgments |
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This work was supported by grants from the National Institutes of Health (to T. Hirano and H.P. Erickson) and the Human Frontier Science Program (to T. Hirano). A. Losada was supported by the Robertson Research Fund and the Leukemia and Lymphoma Society.
Submitted: 1 November 2001
Revised: 4 December 2001
Accepted: 24 December 2001
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References |
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