The canonical condensin complex (henceforth condensin I) plays an essential role in mitotic chromosome assembly and segregation from yeast to humans. We report here the identification of a second condensin complex (condensin II) from vertebrate cells. Condensins I and II share the same pair of structural maintenance of chromosomes (SMC) subunits but contain different sets of non-SMC subunits. siRNA-mediated depletion of condensin I- or condensin II-specific subunits in HeLa cells produces a distinct, highly characteristic defect in chromosome morphology. Simultaneous depletion of both complexes causes the severest defect. In Xenopus egg extracts, condensin I function is predominant, but lack of condensin II results in the formation of irregularly shaped chromosomes. Condensins I and II show different distributions along the axis of chromosomes assembled in vivo and in vitro. We propose that the two condensin complexes make distinct mechanistic contributions to mitotic chromosome architecture in vertebrate cells.

The structural maintenance of chromosomes (SMC) family is a growing family of chromosomal ATPases. The founding class of SMC protein complexes, condensins, plays a central role in mitotic chromosome condensation. We report here a new class of SMC protein complexes containing XSMC1 and XSMC3, Xenopus homologs of yeast Smc1p and Smc3p, respectively. The protein complexes (termed cohesins) exist as two major forms with sedimentation coefficients of 9S and 14S. 9S cohesin is a heterodimer of XSMC1 and XSMC3, whereas 14S cohesin contains three additional subunits. One of them has been identified as a Xenopus homolog of the Schizosaccharomyces pombe Rad21p implicated in DNA repair and the Saccharomyces cerevisiae Scc1p/Mcd1p implicated in sister chromatid cohesion. 14S cohesin binds to interphase chromatin independently of DNA replication and dissociates from it at the onset of mitosis. Immunodepletion of cohesins during interphase causes defects in sister chromatid cohesion in subsequent mitosis, whereas condensation is unaffected. These results suggest that proper assembly of mitotic chromosomes is regulated by two distinct classes of SMC protein complexes, cohesins and condensins.[Key Words: Sister chromatid cohesion; chromosome condensation; the SMC family; X. laevis; cell-free system] Received March 23, 1998; revised version accepted April 29, 1998. Replication and segregation of the genetic information are two of the most fundamental events in cell reproduction. Following replication, chromosomal DNA undergoes three major structural transitions. First, the linkage of duplicated DNA molecules, termed sister chromatid cohesion, is established during or soon after S phase and is maintained throughout G 2 phase of the cell cycle. Second, at the onset of mitosis, the DNA molecules start to condense, producing metaphase chromosomes consisting of paired sister chromatids. Sister chromatid cohesion at this stage is required for proper chromosome movements known as congression. Third, the linkage between the sister chromatids is dissolved highly synchronously at the metaphase-anaphase transition, allowing the two chromatids to segregate to opposite poles of the mitotic spindle. All of these steps are essential for faithful transmission of chromosomes and thereby must be regulated precisely. Despite recent progress in our understanding of the biochemical basis of cell cycle regulation, surprisingly little is known about the molecular mechanisms underlying the dynamic reorganization of chromosome architecture. Particularly, we have very limited information about structural protein components directly involved in these processes (for review, see Miyazaki and Orr-Weaver 1994;Yanagida 1995;Koshland and Strunnikov 1996).

We report here purification and characterization of chromosome condensation protein complexes (termed condensins) containing XCAP-C and XCAP-E, two Xenopus members of the SMC family. Sucrose density gradient centrifugation reveals two major forms of condensins. The 8S form is a heterodimer of XCAP-C and XCAP-E, whereas the 13S form contains three additional subunits. One of them is identified as a homolog of the Drosophila Barren protein whose mutation shows a defect in chromosome segregation. Chromosomal targeting of condensins is mitosis-specific and is independent of topoisomerase IIalpha. 13S condensin is required for condensation, as demonstrated by immunodepletion and rescue experiments. Our results suggest that the condensin complexes represent the most abundant structural components of mitotic chromosomes and play a central role in driving chromosome condensation.

Two different condensin complexes make distinct contributions to metaphase chromosome architecture in vertebrate cells. We show here that the spatial and temporal distributions of condensins I and II are differentially regulated during the cell cycle in HeLa cells. Condensin II is predominantly nuclear during interphase and contributes to early stages of chromosome assembly in prophase. In contrast, condensin I is sequestered in the cytoplasm from interphase through prophase and gains access to chromosomes only after the nuclear envelope breaks down in prometaphase. The two complexes alternate along the axis of metaphase chromatids, but they are arranged into a unique geometry at the centromere/kinetochore region, with condensin II enriched near the inner kinetochore plate. This region-specific distribution of condensins I and II is severely disrupted upon depletion of Aurora B, although their association with the chromosome arm is not. Depletion of condensin subunits causes defects in kinetochore structure and function, leading to aberrant chromosome alignment and segregation. Our results suggest that the two condensin complexes act sequentially to initiate the assembly of mitotic chromosomes and that their specialized distribution at the centromere/kinetochore region may play a crucial role in placing sister kinetochores into the back-to-back orientation. INTRODUCTIONThe faithful segregation of duplicated genetic information into two daughter cells is central to cell proliferation. In eukaryotic cells, amorphous interphase chromatin is converted into individual chromosomes composed of a pair of cylindrical structures (sister chromatids) by metaphase. This process is followed by synchronous segregation of sister chromatids that initiates at the onset of anaphase. During the past decade, a variety of approaches have been combined to demonstrate that a large protein complex called condensin is one of the key regulators of chromosome behavior during mitosis (reviewed by Nasmyth, 2002;Swedlow and Hirano, 2003).The condensin complex is composed of five subunits that are widely conserved among eukaryotic organisms from yeast to humans Sutani et al., 1999;Freeman et al., 2000;Schmiesing et al., 2000;Kimura et al., 2001). The two core subunits of condensin (CAP-E/SMC2 and CAP-C/SMC4) belong to a large family of chromosomal ATPases known as the structural maintenance of chromosomes (SMC) family. The SMC proteins participate in many aspects of higher order chromosome dynamics that include not only chromosome condensation but also sister chromatid cohesion and recombinational repair (reviewed by Hirano, 2002;Jessberger, 2002;Hagstrom and Meyer, 2003). The remaining subunits (CAP-D2, -G, and -H) are not related with SMCs, and share structural motifs with some components involved in cohesion (Neuwald and Hirano, 2000;Schleiffer et al., 2003). The holo-complex of condensin introduces positive superhelical tension into DNA in an ATP hydrolysis-dependent manner in vitro (Kimura and Hirano, 1997;Bazett-Jones et al., 2002), and ...

Structural maintenance of chromosomes (SMC) proteins are ubiquitous in organisms from bacteria to humans, and function as core components of the condensin and cohesin complexes in eukaryotes. SMC proteins adopt a V-shaped structure with two long arms, each of which has an ATP-binding head domain at the distal end. It is important to understand how these uniquely designed protein machines interact with DNA strands and how such interactions are modulated by the ATP-binding and -hydrolysis cycle. An emerging idea is that SMC proteins use a diverse array of intramolecular and intermolecular protein-protein interactions to actively fold, tether and manipulate DNA strands.