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REVIEW

The ABCs of SMC proteins: two-armed for chromosome condensation, cohesion, and repair

Tatsuya Hirano

Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA

The first draft of the human genome sequence was re- structurally similar, protein complexes termed conden- ported a year ago. It may be a good time to remind our- sin and , respectively. At the heart of the two selves that the genetic information encoded in the protein complexeslie membersof a family of chromo- ∼3000-Mb sequence is stored not only in the public or somal ATPases, the structural maintenance of chromo- private databases but also in the tiny space of the cell somes (SMC) family. Equally intriguing, SMC proteins nucleus. The total length of human genomic DNA, are found in most, if not all, bacterial and archaeal spe- which resides in 23 chromosomes, reaches approxi- cies, implicating that their fundamental contribution to mately one meter. It isby no meansa simpletaskto fold chromosome dynamics started even before the acquisi- up the long DNA moleculesand package them within a tion of histonesduringevolution. The goal of thisreview cell nucleuswhosediameter isonly ∼10 µm. Even more article is to discuss the current understanding of higher- strikingisthat the DNA moleculesare faithfully dupli- order chromosome dynamics with an emphasis on the cated and segregated into two daughter cells in an ex- role of SMC proteins. I start with the basic description tremely limited space. Although more than 100 years and classification of SMC proteins and then summarize have passed since Walther Flemming first described the emerging information on the diverse chromosomal func- dynamic behavior of chromosomes (or mitosis) during tions supported by SMC proteins. Finally, I discuss the cell division,it remainshighly mysterioushowthisre- mechanistic aspects of bacterial and eukaryotic SMC markable process of chromosome segregation is achieved proteinsand try to make an integrated picture of their at a mechanistic level. From a cytological point of view, seemingly different actions. two dramatic eventsoccur on chromosomesduringmi- tosis. The first one is the conversion of an amorphous mass of interphase into a discrete set of rod- Basic architecture and classification of SMC proteins shaped chromosomes (chromosome condensation), which occurs from prophase to metaphase (Koshland and The primary structure of SMC proteins, which is shared Strunnikov 1996; Hirano 2000). The second is the split- from bacteria to humans, consists of five distinct do- ting of chromosomes into two halves, which takes place mains(Fig. 1A). Two nucleotide-binding motifs,the highly synchronously at the onset of anaphase (Dej and Walker A and Walker B motifs, are located in the highly Orr-Weaver 2000; Nasmyth et al. 2000). As a crucial pre- conserved N-terminal and C-terminal domains, respec- requisite for these events, duplicated chromosomes (sis- tively. The central domain iscomposedof a moderately ter chromatids) must be held together immediately after conserved “hinge” sequence that is flanked by two long coiled-coil motifs.SMC proteinsform homodimersor DNA replication in S phase and throughout G2 phase. The importance of this process (sister chromatid cohe- heterodimers. An electron microscopy (EM) study of a sion) has been fully appreciated only recently because bacterial SMC homodimer showed that the coiled-coil the pairing of sister chromatids cannot be visualized by motifsare arranged in an antiparallel fashionto make a classical cytology before chromosomes condense in early two-armed, symmetrical structure (Fig. 1B; Melby et al. mitosis. Recent genetic and biochemical studies have 1998). The hydrodynamic propertiesof the SMC dimer begun to shed light on the molecular mechanisms un- are consistent with the idea that the central hinge is derlying cohesion, condensation, and separation of chro- actually flexible and allowsopening and closingof the mosomes during the mitotic cell cycle. One of the un- two arms(Hirano et al. 2001). Thisantiparallel configu- expected findingsisthat chromosomecondensationand ration predictsthat the N-terminal and C-terminal do- sister chromatid cohesion are regulated by distinct, yet mains associate with each other to assemble a globular structure at each end of an SMC dimer. A recent crys- tallographic study has confirmed the formation of this E-MAIL [email protected]; FAX (516) 367-8815. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ catalytic domain in which the Walker A and Walker B gad.955102. motifsmake closecontact (Lowe et al. 2001). Site-di-

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Hirano

Figure 1. Basic architecture of SMC pro- teins. (A) Primary structure of SMC pro- teins. The SMC monomer is a large poly- peptide (between 1000 and 1400 amino acids). The N-terminal (∼160 amino acids) and C-terminal (∼150 amino acids) do- mainsare highly conserved,and contain the nucleotide-binding Walker A and Walker B motifs, respectively. The central domain iscomposedof two long coiled-coil regions(between 300 and 350 amino acids) and a nonhelical hinge sequence (∼200 amino acids). (B) A rotary shadowing image of the Bacillus subtilis SMC ho- modimer (reproduced from J. Cell Biol., 1998, 142: 1595–1604, by copyright per- mission of The Rockefeller University Press). (C) Two modelsfor dimerization of SMC proteins. Coiled-coil interactions between two different subunits may mediate dimerization (left). Alternatively, two self-folded subunits may dimerize by a hinge-mediated interaction (right). Note that, in both cases, the two arms are composed of antiparallel coiled coils. rected mutagenesishasshownthat both motifscontrib- Gram-negative bacteria including lack ute to ATP binding and hydrolysis (Hirano et al. 2001). SMC proteins, a gene product called MukB plays an Despite the progress in our understanding of the archi- analogouscellular function to that of SMCs(for review, tecture of SMC proteins, it remains to be determined see Hiraga 2000). In eukaryotes, at least six members how two polypeptidesare folded to make an SMC dimer. of the SMC are found in individual or- Two models have been proposed so far. First, dimeriza- ganisms. Because each of them has a specific partner tion may be mediated by coiled-coil interactionsbe- with which to form an SMC heterodimer, eukaryotic tween the two different subunits (Fig. 1C, left; Melby et SMC heterodimers can be classified into three distinct al. 1998). Alternatively, the two subunits may be self- groups: SMC1–SMC3, SMC2–SMC4, and SMC5–SMC6 folded to form two separate coiled-coil rods, which, in (Table 1). These heterodimers further associate with dif- turn, dimerize by a hinge-mediated interaction (Fig. 1C, ferent sets of non-SMC subunits to assemble fully func- right; Hirano et al. 2001). It should be noted that con- tional SMC holocomplexes. Both SMC and non-SMC ventional EM does not distinguish between the two subunits appear to contribute to the acquisition of dis- modelsbecausethey predict a virtually identical archi- tinct biochemical and cellular functionsof different tecture of the coiled-coil arms. Further analysis is re- SMC holocomplexes. quired to clarify this important issue. Most of the bacterial and archaeal genomes contain a single smc gene. The minimal functional unit of the SMC2–SMC4: compacting chromosomes gene productsislikely to be a homodimer, ashasbeen and mitotic chromosome condensation shown for the SMC protein from the Gram-positive bacterium Bacillus subtilis (Fig. 2, left; Hirano and Hi- The holocomplex of condensin (also called 13S conden- rano 1998; Melby et al. 1998). Although a subclass of sin) is composed of two SMC subunits (SMC2/CAP-E

Figure 2. Subunit organization of a bacterial SMC protein (BsSMC), and eukaryotic condensin and cohesin complexes. SMC dimers are shown by the V-shaped, two-armed structures. The positions of the non-SMC subunits are arbitrary. Proteins with HEAT repeats are shown in red, and other non-SMC subunits are shown in yellow. The HEAT proteins Scc2 and Pds5 cooperate with cohesin to support sister chromatid cohesion although neither of them is a stoichiometric subunit of the cohesin complex.

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SMC ATPases and chromosome dynamics

Table 1. Components of eukaryotic SMC protein complexes

Subunits S. cerevisiae S. pombe C. elegans D. melanogaster A. thaliana X. laevis H. sapiens

Condensin SMC2 Smc2 Cut14 MIX-1 DmSMC2 BAB11491 XCAP-E hCAP-E SMC4 Smc4 Cut3 F35G12.8 DmSMC4/gluon BAB10693 XCAP-C hCAP-C non-SMC Ycs4 Cnd1 ? CG1911 CAB72176 XCAP-D2/Eg7 hCAP-D2/CNAP1 non-SMC Ycs5/Ycg1 Cnd3 ? CG17054 BAB08309 XCAP-G hCAP-G non-SMC Brn1 Cnd2 ? Barren AAC25941 XCAP-H hCAP-H SMC4 variant — — DPY-27 — — — —

Cohesin SMC1 Smc1 Psm1 F28B34.7 DmSMC1 CAB77587 XSMC1 hSMC1␣ SMC3 Smc3 Psm3 Y47D3A DmSMC3/Cap AAD26882 XSMC3 hSMC3 non-SMC Scc1/Mcd1 Rad21 COH-1,2,3a DmRAD21 >3 homologsXRAD21 hRAD21 non-SMC Scc3 Psc3b F18E2.3 DmSA CAB45374 XSA1 and XSA2 hSA1 and hSA2 SMC1␤ (meiotic) — — — — — ? hSMC1␤ non-SMC (meiotic) Rec8 Rec8 REC-8 ? SYN1/DIF1 ? Rec8 non-SMC (meiotic) — Rec11 ? CG13916? ? ? STAG3/hSA3

SMC5-6 complexc SMC5 YOL034w Spr18 C27A2.1 CG7783 CAC01791(MSS2) AW638169 (est) hSMC5 SMC6 Rhc18 Rad18 C23H4.6 CG5524 MIM BG160113 (est)? hSMC6 aCOH-2 and COH-3 may have meiotic rolesin the germ line (Pasierbeket al. 2001). bPsc3 is not tightly associated with the other cohesion subunits in S. pombe (Tomonaga et al. 2000). cThiscomplex containsother subunitswhoseidentitiesremain to be determined (Fousteriand Lehman 2000).

and SMC4/CAP-C) and three non-SMC subunits (CAP- sizing the functional importance of the five-subunit ho- D2, CAP-G, and CAP-H; Fig. 2, center). Thisfive-subunit locomplex (Kimura and Hirano 2000). Genetic studies of complex wasoriginally identified in Xenopus laevis (Hi- the condensin components have been reported from rano et al. 1997) and subsequently found in different or- many organisms including S. cerevisiae (Strunnikov et ganisms including Schizosaccharomyces pombe (Sutani al. 1995; Freeman et al. 2000; Lavoie et al. 2000; Ous- et al. 1999), (Freeman et al. penski et al. 2000), S. pombe (Saka et al. 1994; Sutani et 2000), and Homo sapiens (Table 1; Schmiesing et al. al. 1999), Caenorhabditis elegans (Lieb et al. 1998), and 2000; Kimura et al. 2001). Two of the non-SMC subunits, (Bhat et al. 1996; Steffensen et CAP-D2 and CAP-G, share a structural motif called the al. 2001). Each one of the five subunits is essential for HEAT repeats(Neuwald and Hirano 2000). The HEAT cell viability in yeast, and this is most likely the case in repeatsare tandem repeatsof an ␣-helical structural all organisms. One of the most prominent phenotypes unit that create a protein-recognition interface with an commonly observed in the condensin mutants is a se- extended solenoidal shape (for review, see Kobe and vere defect in chromosome segregation during anaphase. Kajava 2000). They have been found in a number of pro- The massofchromosomesispulledapart by the mitotic teins with diverse functions, including nuclear trans- spindle, but they fail to segregate properly, exhibiting the port (importin ␤) and transcriptional control (TAF-172/ so-called anaphase bridges. This phenotype is similar, if Mot1). Interestingly, Scc2/Mis4 and Pds5/BimD/Spo76, not identical, to that observed in mutants defective in two gene products genetically implicated in sister chro- topoisomerase II, consistent with the idea that one im- matid cohesion, also contain HEAT repeats (Neuwald portant function of condensin-mediated compaction is and Hirano 2000; Panizza et al. 2000). Although neither to facilitate the resolution of sister chromatids catalyzed of them is a stoichiometric subunit of the cohesin by topoisomerase II (Koshland and Strunnikov 1996; Hi- complex, they cooperate with cohesin to establish and rano 2000). maintain sister chromatid cohesion, further emphasiz- The exact mechanism by which the condensin com- ing the structural (and possibly functional) similarity be- plex contributes to chromosome condensation remains tween the condensation and cohesion machineries (see to be determined. In Xenopus egg extracts, a fluffy and below). unresolved mass of chromatin is produced in the absence In Xenopus egg cell-free extracts, the condensin com- of condensin (Hirano et al. 1997). This phenotype is plex binds to chromosomes in a mitosis-specific manner, clearly distinct from that observed in topoisomerase II- and is required for the establishment and maintenance of depleted extracts(Hirano and Mitchison1993), empha- chromosome condensation (Hirano and Mitchison 1994; sizing the distinct mechanistic contributions of conden- Hirano et al. 1997). Neither the SMC heterodimer nor sin and topoisomerase II to chromosome assembly in the non-SMC subcomplex alone is able to induce chro- vitro. Abnormal chromosome condensation is also a mosome condensation in the cell-free extracts, empha- common phenotype observed in vivo in many condensin

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Hirano mutants, but the extent of condensation defect varies Histone H3 phosphorylation and chromosome between different mutants and different organisms. In condensation Drosophila SMC4 mutants, for example, the shortening of the longitudinal axisof chromosomesisapparently Several different mechanisms have been shown to regu- normal, resulting in the formation of “dumpy” chromo- late condensin functions in vitro and in vivo (Kimura et somes with unresolved sister chromatids (Steffensen et al. 1998; Collaset al. 1999; Sutani et al. 1999; Steen et al. al. 2001). It remainsto be determined whether thisphe- 2000; Kimura et al. 2001). These include enzymatic ac- notype isspecificto the mutationsin the SMC4 subunit tivation of condensin in Xenopus and humans, and mi- or whether the residual level of SMC4 activity in the tosis-specific nuclear transport in S. pombe. Impor- mutant is sufficient to support the axial shortening of tantly, both of the seemingly different levels of regula- chromosomes. In the future, systematic phenotypic tion involve direct phosphorylation of condensin analyses of different mutants should address the specific subunits by the master mitotic kinase cdc2. rolesof individual subunitsinvivo. Reconstitutionof In thisreview, I focuson the potential role of histone subcomplexes in vitro and their functional assessment H3 phosphorylation in condensin recruitment and chro- in Xenopus egg extractsshouldprovide complementary mosome condensation. The N-terminal tail of histone information. H3 is phosphorylated at serine 10, highly coincidently with the onset of mitotic chromosome condensation. In Tetrahymena thermophila, substitution of the serine Condensin and global gene regulation residue with alanine (S10A) affects chromosome conden- In addition to their essential contribution to mitotic sation and segregation (Wei et al. 1999). Recent evidence chromosome condensation and segregation, the conden- suggests that aurora B (also known as Ipl1 in S. cerevisiae sin subunits play important functions at non-mitotic and AIR-2 in C. elegans) islikely to be the major kinase stages of the cell cycle. The best-characterized example that is responsible for this specific phosphorylation (Hsu for such functions is dosage compensation in C. elegans et al. 2000; Spelioteset al. 2000). RNA interference (for review, see Meyer 2000). The SMC2 ortholog MIX-1 (RNAi) experimentsin Drosophila also support this con- associates with DPY-27 (a variant form of SMC4) to form clusion (Adams et al. 2001b; Giet and Glover 2001). In a dosage compensation complex along with additional Aspergillus nidulans, another kinase known as NimA subunits including DPY-26 and DPY-28 (Lieb et al. actsasa histoneH3 kinase(De Souza et al. 2000). Mu- 1998). Thiscomplex isspecificallytargeted to both X tation or depletion of these H3 kinases causes defects in chromosomesofhermaphroditesto repressthelevel of multiple events in mitosis including chromosome segre- transcription by half. MIX-1 is also a component of the gation and cytokinesis(Speliotesetal. 2000; Adamset al. condensin complex that participates in mitotic chromo- 2001b; Giet and Glover 2001). some condensation and segregation. It is of great interest How doesa lossofH3 phosphorylationaffect chromo- to determine whether the dosage compensation machin- some segregation? A popular model is that the modifi- ery accomplishes chromosome-wide gene repression by cation may send a signal to initiate chromosome con- using the same mechanism that drives chromosome con- densation. The phosphorylated tail of histone H3 could densation in mitosis. function asa receptor that recruitschromosomeconden- In Drosophila, the polycomb group (PcG) proteinsact sation proteins such as the condensin complex (Wei et al. on specialized cis-elements (polycomb response ele- 1999). Consistent with this idea, a non-SMC subunit of ments, PRE) to maintain the transcriptionally repressed condensin, Barren, is not properly targeted to chromo- state of homeotic genes. A recent study using chromatin someswhenaurora B isdepleted by RNAi in Drosophila immunoprecipitation assays has revealed that topoisom- (Giet and Glover 2001). It isunclear, however, whether erase II and the condensin subunit Barren/CAP-H colo- thisisa direct consequenceof the failure of H3 phos- calize on DNA sequencesincludingthe PREsin the bi- phorylation or an indirect effect of other problems thorax complex (Lupo et al. 2001). Moreover, genetic caused by the absence of aurora B activity. In a purified experimentshave shownthat Barren isrequired for system, for example, phosphorylation of histone H3 at gene silencing mediated by one of the PREs, Fab-7. Thus, serine 10 has little impact on the interaction between the condensin subunit and PcG proteins appear to coop- condensin and nucleosomes (Kimura and Hirano 2000). erate to maintain the silenced state of gene expression, Moreover, in Xenopus egg extracts, condensin can inter- possibly by assembling condensed heterochromatin-like act with “tailless” nucleosomes (de la Barre et al. 2000), structures. and artificial induction of H3 phosphorylation is not suf- In S. cerevisiae, condensin concentrates in the ficient to recruit condensin to chromosomes (Murnion et rDNA region during mitosis. Interestingly, this bind- al. 2001). Finally and most importantly, a recent study ing to rDNA persists in interphase, implying that shows that neither chromosome condensation nor chro- condensin may have a specialized function in organiz- mosomal targeting of condensin is compromised when ing thishighly repetitive locuswith propertiesof H3 phosphorylation is drastically reduced by depletion heterochromatin (Freeman et al. 2000). An apparent of aurora B from the extracts(MacCallum et al. 2002). enrichment of condensin subunits in the Thus, the exact role of this modification in chromosome hasalsobeen reported in human cells(Cabello et al. dynamics remains elusive. In addition to serine 10, ser- 2001). ine 28 of histone H3 is phosphorylated in a mitosis-spe-

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SMC ATPases and chromosome dynamics cific manner (Goto et al. 1999). Unlike Tetrahymena, data are all intriguing but fragmentary. Future work single or double mutations in these phosphorylation should address how the SUMO (and ubiquitin) pathway sites in S. cerevisiae cause no detectable defects in chro- might directly (or indirectly) affect the condensation ma- mosome segregation (Hsu et al. 2000), providing an ad- chinery in these organisms. ditional complexity to thisproblem. It ispossiblethat combinatorial modificationsof different histonetailsare important for regulating chromosome behavior in mito- SMC1–SMC3: holding chromatids together sis, as is the case in transcriptional regulation (for re- Cohesin and its interacting proteins view, see Stahl and Allis 2000). A recent series of biochemical, cytological, and genetic The cohesin complex consists of the heterodimer of studies strongly suggests that aurora B functions to- SMC1 and SMC3 and at least two non-SMC subunits gether with inner centromere protein (INCENP) in a pro- (Scc1/Mcd1/RAD21 and Scc3/SAs; Fig. 2, right). The tein complex (Adamset al. 2000, 2001a; Kaitna et al. subunits of cohesin were systematically identified in S. 2000) that may also contain a small protein called sur- cerevisiae by a genetic screen for mutants that display vivin/BIR-1 (Spelioteset al. 2000; Uren et al. 2000; Mor- premature separation of sister chromatids (Michaelis et ishita et al. 2001; Wheatley et al. 2001). These three pro- al. 1997). Some of them were identified independently by teins are collectively referred to as chromosomal passen- screensformutantsthat affect proper segregationof mi- gerson the basisoftheir dynamic and characteristic totic chromosomes (Strunnikov et al. 1993; Guacci et al. localization during mitosis. The chromosomal passen- 1997). The protein complex containing the correspond- gers are associated with chromosome arms during the ing gene productswasfound in Xenopus (Losada et al. early stages of mitosis and accumulate progressively at 1998), and later in other organisms including S. cerevi- inner centromeresby metaphase.They leave chromo- siae (Toth et al. 1999), S. pombe (Tomonaga et al. 2000), somes in anaphase, redistributing to the spindle midzone and humans(Table 1; Losadaet al. 2000; Sumara et al. and equatorial cortex. Both INCENP and survivin/BIR-1 2000). In higher eukaryotic cells, several different iso- are required for the proper localization of aurora B. Given types are found in each of the two non-SMC subunits. thisdynamic behavior, it isnot surprisingto find that For example, vertebrate cellshave three Scc3/SA ho- the loss-of-function mutation of this class of proteins mologs. SA1 and SA2 form distinct complexes termed causes highly complex phenotypes. Conceivably, his- cohesinSA1 and cohesinSA2 in mitotic cells(Losadaet al. tone H3 is only one of the many substrates that are phos- 2000; Sumara et al. 2000), whereasthe third homolog, phorylated by aurora B during mitosis, and identifica- SA3/STAG3, has a -specific function (Pezzi et al. tion of nonhistone substrates is one of the important 2000; see below). The C. elegans genome hasfour Scc1 future directions. It is unknown whether condensin sub- homologswhosefunctionsare differentially regulated units are among the substrates of the aurora B–INCENP during development (Pasierbek et al. 2001). complex. Chromosomal binding sites of cohesin have been mapped in S. cerevisiae by chromatin immunoprecipita- tion assays. Cohesin associates with specific regions The SUMO pathway and chromosome condensation: near centromeresand along chromosomearmswith a a potential link? preference for AT-rich sequences. In the arms, cohesin SUMO (small ubiquitin-related modifier) is a conserved distributes with a periodicity of ∼15 kb, asjudged by a ubiquitin-like small protein that is covalently attach- chromosome-wide hybridization approach (Blat and ed to other proteinsto modulate their functions(for re- Kleckner 1999), or of ∼9 kb, asrevealed by high-resolu- view, see Melchior 2000). Recent studies point out a po- tion chromosome walking (Laloraya et al. 2000). There is tential link between this posttranslational modification no apparent correlation between replication originsand pathway and chromosome condensation. In S. cerevi- the cohesin-binding sites. The association of cohesin siae, the temperature sensitivity of a condensin mutant, with centromeresrequiresfunctional kinetochore pro- , is suppressed by overexpression of Smt4, a prote- teins(Tanaka et al. 1999) and increasesinmitotically ase that possesses SUMO-cleavage activity (Strunnikov arrested cells (Laloraya et al. 2000), suggesting that co- et al. 2001). Smt4 is not an essential protein, but its null hesin binding may be regulated differentially at centro- mutation decreases the fidelity of chromosome segrega- meres and chromosome arms. In S. pombe, it hasbeen tion and affects mitosis-specific targeting of condensin shown recently that Swi6, a counterpart of the hetero- to rDNA. The slow-growth phenotype of ⌬ issup- chromatin protein HP1, playsa role in recruiting cohesin pressed by overexpression of Siz1, a protein that pro- specifically at centromeres (Bernard et al. 2001). motesSUMO conjugation in vitro (Johnsonand Gupta In both yeast and Xenopus, the loading of cohesin onto

2001). In Drosophila, mutationsin the Su(var)2-10 locus, chromatin in G1 is functionally separable from the es- which encodes a Siz1 homolog, cause chromosome tablishment of sister chromatid cohesion in S phase transmission defects and abnormal chromosome mor- (Losada et al. 1998; Uhlmann and Nasmyth 1998). In S. phologies(Hari et al. 2001). On the other hand, a muta- cerevisiae, the HEAT protein Scc2 associates with Scc4 tion of a component of the ubiquitin CUL-2 to form a complex required for the loading of cohesin causes defects in chromosome condensation in C. el- onto chromatin (Ciosk et al. 2000). In S. pombe, Mis4 egans (Feng et al. 1999). Thus, the currently available playsa role analogousto Scc2 (Tomonaga et al. 2000).

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Hirano

How thisloading processisachievedisstillunknown. ture efforts should address the biochemical mechanism Another HEAT protein called Pds5 is required for the by which the passage of replication forks directs the con- establishment and maintenance of cohesion in S. cerevi- struction of a physical bridge between newly synthesized siae (Hartman et al. 2000; Panizza et al. 2000). The role DNA strands. It is tempting to speculate that this pro- of thisclassofproteinsin other organisms(Pds5in S. cessaccompaniesaconformational change of the cohe- pombe, BimD in A. nidulans, and Spo76 in Sordaria ma- sin complex or its enzymatic activation. crospora) is far less clear, although several studies have shown that Pds5/BimD interacts genetically and physi- cally with the cohesin complex (Holt and May 1996; Unloading of cohesin and sister chromatid separation Sumara et al. 2000; Tanaka et al. 2001). For example, Pds5 is not essential for mitotic growth in S. pombe under nor- An elegant series of genetic and biochemical experi- mal conditions(Tanaka et al. 2001), nor isBimD in A. mentsin S. cerevisiae has shown that the cysteine pro- nidulans at low temperatures(van Heemstet al. 2001). In tease Esp1 (or separase) cleaves the cohesin subunit Scc1, Sordaria, mutations in Spo76 cause only subtle defects in thereby promoting sister chromatid separation at the on- the mitotic cell cycle, whereasthey displayprominent set of anaphase (Uhlmann et al. 1999, 2000). Phosphory- phenotypes in meiotic chromosome morphogenesis (van lation of Scc1 by the Polo/Cdc5 kinase enhances this Heemst et al. 1999). Interestingly, when Pds5 is deleted, S. cleavage reaction (Alexandru et al. 2001). A similar pombe cellsbecome viable even in the absenceof the co- schemeislikely to operate in other eukaryotesaswell. A hesion protein Eso1 (see below) that is otherwise essential small fraction of Scc1/RAD21 is cleaved during anaphase for mitotic growth (Tanaka et al. 2001). Further work will in S. pombe (Tomonaga et al. 2000) and human cells be required to determine the biochemical functionsof (Waizenegger et al. 2000). Moreover, ectopic expression these HEAT proteins and to clarify the seemingly diverse of cleavage-resistant forms of Scc1/RAD21 disturbs mutant phenotypes in different organisms. chromosome segregation in these organisms (Tomonaga et al. 2000; Hauf et al. 2001), ashasbeen shownin S. cerevisiae (Uhlmann et al. 1999). Functional coupling between DNA replication Despite the conserved mechanism involving cohesin and sister chromatid cohesion cleavage in anaphase, a striking difference exists in the Sister chromatid cohesion is established during S phase. regulation of sister chromatid cohesion between S. cer- Recent studies have begun to address the question of evisiae and higher eukaryotic cells. Unlike in yeast, how cohesion factors functionally interact with the most cohesin (∼95%) dissociates from chromatin during DNA replication machinery. In S. cerevisiae, Ctf7/Eco1 prophase, far before the onset of anaphase, in metazoan is required for the establishment but not for the mainte- cells(Losadaet al. 1998, 2000; Sumara et al. 2000; Wai- nance of cohesion, and genetically interacts with the zenegger et al. 2000). The exact mechanism of this pro- sliding clamp PCNA (Pol30) and its putative loader phase dissociation is unknown, although there is an in- Ctf18 (Skibbenset al. 1999; Toth et al. 1999). The bind- dication that phosphorylation of the cohesin subunit SA/ ing of cohesin to chromatin is apparently normal in ctf7/ Scc3 might be part of it (Losada et al. 2000). The eco1 mutants. Intriguingly, Ctf7/Eco1-like sequences are remaining ∼5% of cohesin is apparently enriched in the present in highly variable forms among different organ- centromere-proximal region under mitotically arrested isms. For example, S. pombe Eso1 is composed of an conditions(Waizenegger et al. 2000; Warren et al. 2000; Eco1-related domain essential for cohesion, and a DNA Hoque and Ishikawa 2001), leading to the proposal that polymerase ␩-related domain implicated in translesion cohesin dissociates first from chromosome arms during DNA synthesis (Tanaka et al. 2000). The Drosophila ho- prophase and then from centromeres in anaphase. It re- molog displays a different chimeric organization. More mainsto be determined, however, exactly how the tem- recent studies in S. cerevisiae have provided additional poral and spatial dissociation of cohesin is regulated in evidence for the direct link between the DNA replica- normal mitosis, in which chromosome arms are also tion machinery and sister chromatid cohesion. First, a held together until the onset of anaphase. novel DNA polymerase activity (Pol ␴, renamed from Why does cohesin dissociate from chromatin during Pol ␬) is associated with Trf4, a protein involved in co- prophase in metazoan cells? It is reasonable to speculate hesion (Wang et al. 2000). Second, the establishment of that thispartial lossofcohesionisa prerequisiteto the proper cohesion requires an alternative clamp loader initiation of condensin-mediated condensation in pro- containing Ctf18, Ctf8, and Dcc1, aswell asanother pro- phase. Conceivably, a high density of cohesin on a chro- tein, Ctf4, that physically interacts with the catalytic mosome arm limits the size of chromatin loops and subunit of DNA polymerase ␣ (Hanna et al. 2001; Mayer thereby constrains the action of condensin in folding and et al. 2001). On the basis of these results, a polymerase compacting each loop. On the basisofthisidea, it has switching model has been proposed in which these rep- been proposed that the shape of the metaphase chromo- lication factorsmay be dedicated specificallyto replicate some is determined by a precise balance between the cohesin-associated regions of chromosomal DNA. cohesion and condensation machineries (Losada and Hi- Therefore, the replication-coupled establishment of co- rano 2001b). In higher eukaryoteswith large genomes, hesion appears far more complex than previously antic- cohesion along chromosome arms must be released to ipated, even in a simple organism like S. cerevisiae. Fu- allow efficient condensation, and loosening of arm cohe-

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SMC ATPases and chromosome dynamics sion is counterbalanced by an increased cohesion around along the AEsin pachytene, the integrity of thiscore centromeres. structure is apparently intact even in the absence of the AEs(Pelttari et al. 2001). It shouldbe noted that the reverse may not be the case: cohesin function is required Meiosis-specific cohesin components for proper formation of the AEsat leastin S. cerevisiae Given the fundamental role of cohesin in sister chroma- (Klein et al. 1999). Intriguingly, the cohesin core, with- tid cohesion during mitosis, it is not surprising to find out the AEs, can recruit recombination proteins and pro- that the cohesin subunits and other cohesion factors play mote synapsis between homologous chromosomes (Pelt- vital roles in meiotic chromosome pairing and segrega- tari et al. 2001). Thus, the new study leaves a number of tion. Emerging lines of evidence suggest that eukaryotes fundamental questions on the structural and functional have evolved meiosis-specific cohesin components to basis of meiotic chromosome pairing, recombination, modify the preexisting mitotic program. The best stud- and segregation. ied example of suchcomponentsisRec8, which hasa similarity to the cohesin subunit Scc1/RAD21 (Micha- SMC5–SMC6: linking DNA repair and checkpoint elis et al. 1997) and is conserved from yeast to humans responses (Table 1; Parisi et al. 1999). In S. cerevisiae, Rec8 and Phylogenetic analysesrevealthat eukaryotic cellshave Smc3 colocalize to chromosomal cores in prophase I and two additional membersof the SMC family (e.g., Cobbe dissociate from chromosome arms in metaphase I, but and Heck 2000). Recent biochemical studies in S. pombe remain bound to centromeresuntil metaphaseII (Klein and human cells have shown that these two proteins et al. 1999). Rec8 function is essential for cohesion, for- (now called SMC5 and SMC6) form a protein complex mation of axial elements(AEs),and recombination. The along with additional subunits whose identity remains cleavage of Rec8 by separase in anaphase I is necessary to be established (Fousteri and Lehmann 2000; Taylor et for the release of arm cohesion and thereby for the dis- al. 2001). SMC6 wasoriginally identified asthe gene junction of homologouschromosomes(Buonomoet al. product of rad18 in S. pombe, whose mutation causes 2000). In S. pombe, it hasbeen shownthat Rec8 isre- hypersensitivity to both UV and ␥ radiation (Lehmann et quired to establish reductional chromosome segregation al. 1995). Unlike other Rad gene products, Rad18/SMC6 in meiosis I (Watanabe and Nurse 1999), and that this is essential for mitotic growth in S. pombe, and thisis function isprimed during the premeiotic S phaseat the also the case with SMC5 (also known as Spr18; Fousteri inner centromeric region (Watanabe et al. 2001). Rec8 and Lehmann 2000). Rad18/SMC6 isrequired to main- homologsinvolved in meiotic chromosomepairing and tain a checkpoint arrest after DNA damage, and it ge- disjunction have also been characterized in Arabidopsis netically interacts with Brc1, a nonessential protein that thaliana (Bai et al. 1999; Bhatt et al. 1999) and C. elegans shares BRCT domains with the breast cancer suscepti- (Pasierbek et al. 2001). bility gene product BRCA1 (Verkade et al. 1999). More- The S. pombe genome hastwo Scc3/SA-like se- over, rad18 issyntheticallylethal with a mutant of to- quences, and one of them, Rec11, is involved in meiotic poisomerase II, but not with mutants of condensin or cohesion and recombination (Table 1; Krawchuk et al. cohesin. The SMC5–SMC6 protein complex is therefore 1999). In mammalian cells, a similar protein, STAG3 likely to play a role in higher-order chromosome organi- (also known as SA3), has been implicated in sister chro- zation, independently of condensation and cohesion, matid arm cohesion during meiosis I (Prieto et al. 2001). that is essential for genomic integrity and DNA damage In S. cerevisiae, there isno meiotic counterpart that be- responses. In mammals, SMC5 and SMC6 are highly ex- longsto thisclassofcohesionfactors. pressed in the testis and associate with the X–Y chromo- More recently, a meiosis-specific SMC protein has some pair in the late stage of meiotic prophase (Taylor et been reported in mammalian cells(Revenkova et al. al. 2001), implicating their additional functions in meiosis. 2001). Thisnewestmember of the SMC family ismost In Arabidopsis, a mutant of an SMC6 homolog (called closely related with SMC1 (therefore named SMC1␤) and mim) is hypersensitive to a variety of DNA-damaging isnot found in the genome of yeast, Drosophila, C. el- agentsand isdefective in intrachromosomalhomolo- egans,orArabidopsis. It associates with SMC3 but not gousrecombination in somaticcells(Mengisteet al. with the canonical SMC1 (or SMC1␣). SMC1␣ isloosely 1999). Surprisingly, the homozygous mim plantsdevelop associated, in a punctate pattern, with the AEs of the normally, providing the first example of an eukaryotic synaptonemal complexes (SCs) at the pachytene stage SMC mutant that doesnot affect the viability of an (Eijpe et al. 2000), whereasSMC1␤ ismore tightly and organism. It has been proposed that MIM plays an uniformly distributed along the AEs. Importantly, al- active role in homologousrecombination by increasing though SMC1␣ dissociates from the chromatin in late accessibility of chromosomal DNA to the recombination prophase I, SMC1␤ remainsat the centromeresuntil machinery. metaphaseII. Thisbehavior predictsthat SMC1 ␤, not SMC1␣, is responsible for maintaining cohesion be- Primordial SMCs: illuminating the evolution tween sister centromeres in meiosis II. of chromosome dynamics Another recent study in mammalian meiotic cells has provided additional insight into meiotic chromosome A requirement for SMC proteinsin bacterial chromo- structure. Although cohesin forms a chromosomal core some partitioning has been shown in B. subtilis and

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Hirano

Caulobacter crescentus. B. subtilis smc null mutantsare E. coli support the speculation that a cohesion process temperature-sensitive in rich growth medium and show exists in bacterial cells (Hiraga et al. 2000; Ohsumi et al. multiple phenotypes at permissive conditions, including 2001). This putative cohesion may be essential for abnormal morphology, mislocalization of the postreplicative repair as suggested in eukaryotic cells origin region, and accumulation of anucleate cells(Brit- (Sjogren and Nasmyth 2001). If SMC/MukB is involved ton et al. 1998; Graumann et al. 1998; Moriya et al. 1998; in this process, then it would further emphasize the Britton and Grossman 1999; Graumann 2000). Similar mechanistic similarity between the bacterial and eu- phenotypesare observedin the null mutant of the smc karyotic chromosome cycle. In eukaryotic cells, the four gene in C. crescentus (Jensen and Shapiro 1999). Few major eventsof the chromosomecycle (duplication, co- anucleate cells are produced under permissive condi- hesion, condensation, and separation) are functionally tionsin thisspecies,however, implying that a cell cycle coordinated with each other, but are temporally sepa- checkpoint operatesto arrestmutant cellsat a predivi- rated and occur at discrete stages of the cell cycle (Fig. sional stage. Increasing lines of recent evidence suggest 3B). In the bacterial chromosome cycle, these events that, in E. coli, MukB actsasthe functional homolog of take place simultaneously in a cell (Fig. 3A). Despite the SMC. Although the primary sequences of MukB and outward differencesin regulation, the mechanisticpar- SMC show a very limited homology, the two proteins do allel between the two systems is obvious. For example, share a remarkably similar two-armed structure, as bacterial SMC proteins may facilitate separation and seg- judged by electron microscopy (Melby et al. 1998). Mu- regation of nucleoidsby pulling and compacting them tant phenotypesof E. coli mukB are almost indistin- into the cell poles(Sawitzke and Austin2000). This guishable from those observed in B. subtilis smc mu- could accompany the loss of putative cohesion, or act in tants(Niki et al. 1991). Unlike the B. subtilis SMC concert with extrusion of nascent DNAs by the replica- (BsSMC) dimer, the MukB dimer associates with two tion machinery (Lemon and Grossman 1998). The analo- other proteins, MukE and MukF, to form a three-subunit gousprocessinthe eukaryotic chromosomecycle is protein complex (Yamazoe et al. 1999). Moreover, mukE metaphase chromosome condensation, in which sister and mukF mutants display similar phenotypes to that of chromatids are partially separated (or resolved) by con- mukB, suggesting that the three gene products act in densin-mediated compaction and the accompanying loss concert in vivo. Although no apparent homolog of MukE of cohesin. The final separation, which is triggered by or MukF isfound in the genome of B. subtilis, it will be the cleavage of cohesin at anaphase, uses eukaryote-spe- important to know whether BsSMC functions together cific machinery, the mitotic spindle. This idea would with loosely associated non-SMC subunits whose struc- explain the ancient origin of SMC-mediated chromo- turesmay be highly divergent from MukE and MukF. some separation/segregation and the apparent lack of the A large number of proteinshave been shownto inter- spindle apparatus in bacterial cells. Further genetic, bio- act genetically with MukB (for review, see Hiraga 2000). chemical, and cell biological studies will be required to A recent important finding isthat mutationsin topoi- test and extend this idea and to enhance our understand- somerase I (topA) suppress the mukB phenotypes(Saw- ing of the evolutionary originsof chromosomaldynam- itzke and Austin 2000), and that mukB mutantsare hy- ics. persensitive to inhibitors of DNA gyrase (Weitao et al. 1999; Sawitzke and Austin 2000). These results suggest that MukB may participate in higher-order chromosome Molecular mechanisms of SMC actions: toward folding by modulating DNA topology, a mechanism a unified view analogous, if not identical, to that proposed for conden- As discussed above, SMC proteins play highly diverse sin-mediated chromosome condensation in eukaryotic functionsin regulating chromosomedynamicsin eu- cells(Kimura and Hirano 1997; Kimura et al. 1999). karyotic cells, including chromosome condensation, sis- The identification and functional characterization of ter chromatid cohesion, recombinational repair, and bacterial SMC (and MukB) proteinshave provided us global gene repression. What do these seemingly differ- with an excellent opportunity to compare and contrast ent chromosomal processes have in common? How do chromosome dynamics between the prokaryotic and eu- SMC proteins support these processes at a mechanistic karyotic systems. For example, it has remained elusive level? How similar and how different are the actions of for decadeshow bacterial nucleoidsmight be organized bacterial and eukaryotic SMC proteins? In this section, at a higher-order level. Thisproblem can now be revis- an attempt is made to answer these questions from a ited with the new idea that bacterial SMC/MukB pro- mechanistic point of view. teins may share a common mechanism of action with the eukaryotic condensin complex. It is important to point out that thisidea, in turn, raisesanotherquestion: The ATP-binding and hydrolysis cycle of SMC proteins does the bacterial chromosome cycle have a process cor- responding to sister chromatid cohesion? If the bacterial An early sequence analysis pointed out that all SMC SMC/MukB protein isthe common ancestorof the eu- proteins share a unique motif (called the signature motif karyotic cohesin and condensin complexes, it could play or the C motif) that ishighly conservedamong members a role in cohesionaswell asin condensation.In fact, the of the ATP-binding cassette (ABC) superfamily (Saitoh et localization and movement of nascent DNA clusters in al. 1994). A recent crystallographic study has shown that

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Figure 3. Rolesof SMC proteinsin the bacterial and eukaryotic chromosome cycle. (A) In bacterial cells, chromosome replica- tion and segregation take place simulta- neously. Chromosome separation and segre- gation are facilitated by a condensin-like function of SMC proteins(magenta). SMC proteinswith a putative cohesin-likefunc- tion (green) may also be involved. Repli- cated and unreplicated chromosomal re- gions are shown in blue and orange, respec- tively. Replication forksare indicated in black. (B) In eukaryotic cells, each chromo- somal event occurs at a discrete stage of the cell cycle. When chromosomal DNA is du- plicated during S phase, sister chromatid co- hesion is established by the action of cohe- sin (green). The linkage between sister chro-

matidsismaintained during G 2 phase, and is partially dissolved by metaphase to allow chromosome condensation mediated by condensin (magenta). This partial separa- tion (or resolution) step is most similar to chromosome partitioning in bacterial cells. The full separation of sister chromatids is triggered at the onset of anaphase and is completed by the action of the mitotic spindle (data not shown). the catalytic domain of an SMC protein, composed of the land and Blight 1999) and the double-strand-break repair N and C termini, indeed displays a protein fold similar to protein Rad50 (for review, see Haber 1998). A common that of the corresponding domains of ABC ATPases (Fig. structural feature of these ABC ATPases is that each 4A; Lowe et al. 2001). Therefore, SMC proteinsbelong to functional complex containstwo catalytic domains(also this large superfamily of ATPases, members of which called nucleotide-binding domains, NBDs). In the case of include numerousABC transporters(forreview, seeHol- ABC transporters, the two NBDs cooperatively modulate

Figure 4. SMC proteins belong to the ABC ATPase superfamily. (A) Crystal structure of an SMC catalytic domain consisting of the N-terminal (orange) and C-terminal (blue) sequences (reproduced from J. Mol. Biol., 2001, 306: 25–35, by copyright permission of Academic Press). Three important motifs, Walker A, Walker B, and ABC signature (or C motif), are indicated. (B) Hypothetical ATP-binding and hydrolysis cycle of SMC proteins. SMC ATPase may act as a composite ATPase, in which hydrolysis of ATP is triggered by the interaction between the two catalytic domains.

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Hirano neighboring transmembrane domains (TMDs) so that dimer interaction is observed and ATP hydrolysis is ac- small molecules (e.g., ions, amino acids, and lipids) are tivated only by the intramolecular mode. When BsSMC actively transported across the cellular membrane. bindsto DNA, ATP promotesa dimer–dimer interac- Rad50 forms a homodimer whose two-armed structure is tion, which, in turn, activatestheir DNA-dependent very similar to that of SMC dimers (Hopfner et al. 2000; ATPase by the intermolecular mode. This bimodal acti- Anderson et al. 2001). Biochemical data suggest that the vation model providesa natural explanation for the two catalytic domainsof ABC proteinsfunctionally in- unique, two-armed structure of SMC proteins, although teract with each other to modulate their ATPase activ- itsphysiologicalsignificancein bacterial cellsneedsto ity. It ishighly controversial,however, how thismight be explored. The model further emphasizes the func- be achieved at the structural level, because most of the tional flexibility and large potential of the unique design protein crystals solved to date are monomeric forms. of this class of ABC ATPases. One important exception isRad50 (Hopfner et al. 2000). The crystal structure of a nucleotide-bound form of Molecular actions of condensin and cohesin Rad50 shows that ATP binding induces the association of the two catalytic domainsand thereby createsa DNA- The bimodal activation model predictsthat opening and binding surface. Two ATP molecules are sandwiched in closing of the coiled-coil arms make fundamental con- the interface of the catalytic domains, and their hydro- tributionsto the actionsof SMC proteins.How can this lysisrequiresaproper interaction of the two catalytic idea be extended to explain the actionsof eukaryotic domains. It remains to be determined to what extent the SMC protein complexes? A recent biochemical study has information deduced from the Rad50 structure may be revealed that purified condensin and cohesin show strik- applicable to the action of SMC ATPases, because the ingly different DNA-binding propertiesin vitro (Losada biochemical activitiesof Rad50 and SMCsare substan- and Hirano 2001a). In a simple gel-shift assay, for ex- tially different. For example, unlike Rad50, neither ATP ample, condensin produces a discrete set of shifted binding nor dimerization of the catalytic domainsises- bands, whereas cohesin induces the formation of large sential for the DNA-binding activity of SMC proteins protein–DNA aggregates. These results are consistent (Hirano and Hirano 1998; Hirano et al. 2001). Despite with our previoushypothesisthatcondensinmight func- these seemingly different functional characters, it would tion as an intramolecular DNA cross-linker that folds a be reasonable to speculate that Rad50 and SMCs share a single DNA molecule, whereas cohesin might act as an common scheme of ATP binding and hydrolysis (Fig. 4B). intermolecular DNA cross-linker that holds two differ- This type of composite ATP- is also found in ent DNA segments together (Hirano 1999). An impor- the mismatch DNA repair protein MutS (Junop et al. tant mechanistic question is how condensin and cohesin 2001), and may represent a widespread feature of an even are able to distinguish between the intramolecular and larger group of ATPases beyond the canonical ABC pro- intermolecular modesof interaction with DNA. One teins. Most recently, the crystal structure of a dimeric possibility is that different conformations of the SMC form of the bacterial ABC transporter MsbA has been subunitsconferthe two different modesof DNA inter- determined (Chang and Roth 2001). The V-shaped ar- actions. For instance, the arms of condensin may prima- rangement of the two transmembranedomainsisremi- rily be closed, and the action of the two catalytic do- niscent of the two-armed structure of SMCs and Rad50, mains of SMC2–SMC4 would be restricted so that they further suggesting a common mechanism of action of the can only bind to contiguousDNA segments(Fig.5C). On ABC ATPases. (It should be added, however, that the the other hand, an open conformation of cohesin’s arms dimer interface deduced from the current MsbA crystal may allow the two catalytic domainsof SMC1–SMC3 to isdifferent from that of Rad50.) bind to two noncontiguous DNA segments. This could further be facilitated or strengthened by the protein–pro- tein interaction between two cohesin complexes (Fig. Bimodal activation model of SMC ATPase 5D). An additional prediction of the bimodal activation What isthe role of the ATP-binding and hydrolysiscycle model isthat the dynamic DNA interactionsof conden- in the actionsof SMC proteins?If the two catalytic do- sin and cohesin may be regulated primarily by the intra- mains of SMC proteins constitute a composite ATPase, molecular and intermolecular modesof ATPasecycle, then the two-armed, symmetrical structure predicts, in respectively (Fig. 5C,D). We suggest that the two eukary- principle, two distinct modes of ATPase activation. otic SMC protein complexesare structurallyand func- First, closing of the arms would trigger ATP hydrolysis tionally differentiated from the prototype of SMC pro- by allowing an interaction between the two catalytic do- teins (e.g., BsSMC). It is of great interest to test whether mainswithin a dimer (intramolecular mode; Fig. 5A). the establishment and dissolution of cohesion is func- Second, opening of the armswould allow the catalytic tionally coupled with the ATP-binding and hydrolysis domainsof one dimer to interact with thoseof a neigh- cycle of the cohesin complex. boring dimer, thereby causing ATP hydrolysis (intermo- The condensin complex actively reconfigures the lecular mode; Fig. 5B). A recent mechanistic analysis of DNA structure by using the energy of ATP hydrolysis in the BsSMC homodimer has provided evidence that both vitro. Two different assays have been used to character- activation modesmay, indeed, be usedby SMC proteins ize these activities. In the presence of topoisomerase I, (Hirano et al. 2001). In the absence of DNA, no dimer– condensin introduces positive supercoils into relaxed

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Figure 5. Dynamic actions of SMC ATPases supported by the two-armed structure. (A,B) Bimodal activation of SMC ATPase. Closing of the armstriggersATP hydrolysisbyallowing the interaction between the two catalytic domainswithin an SMC dimer ( A, intramolecular mode). Opening of the armsallowsthe catalytic domainsof one dimer to interact with thoseof another dimer, which, in turn, activatesATP hydrolysis( B, intermolecular mode). (C,D) Hypothetical actions of condensin and cohesin. (C) Condensin may primarily use the intramolecular ATPase mode to compact a single DNA molecule. (D) Cohesin may use the intermolecular ATPase mode to promote and modulate interactionsbetween two different DNA molecules. circular DNA (Kimura and Hirano 1997; Kimura et al. hypothetical ATP-dependent activities. Candidates for 2001). In the presence of topoisomerase II, condensin such factors may include Scc2/Mis4 and Pds5/BimD/ convertsnicked circular DNA into positivelyknotted Spo76, two HEAT-containing proteinsimplicated in es- forms(Kimura et al. 1999, 2001). Neither of theseactivi- tablishing cohesion in concert with cohesin. Very little tiescan be supportedby the core SMC2–SMC4 dimer isknown at presentabout the biochemical propertiesof alone, suggesting that the non-SMC subunits are ac- the SMC5–SMC6 complex (Fousteri and Lehmann 2000) tively involved in these reactions (Kimura and Hirano or the MukBEF complex (Yamazoe et al. 1999). 2000). Although these activities are compatible with the action of condensin predicted above, a full understanding Future directions of the mechanism requires a combination of structural and biophysical approaches including electron micros- The first genetic study of an SMC protein in yeast was copy and single-molecule manipulations. Much less is published only eight years ago (Strunnikov et al. 1993). known about the molecular action of the cohesin com- Since then, we have witnessed unusually rapid progress plex. In the presence of topoisomerase II, cohesin directs in this research field and enjoyed a very rich harvest, intermolecular catenation of DNA asopposedto intra- which hascompletely changed our view of chromosome molecular knotting promoted by condensin (Losada and dynamics.There isno doubt that SMC proteinsare cen- Hirano 2001a). Thisaction of cohesin,however, doesnot tral to a broad spectrum of higher-order chromosome dy- require ATP, and purified cohesin shows very low, if any, namicsin organismsrangingfrom bacteria to humans. ATPase activity (A. Losada and T. Hirano, unpubl.). One Our present knowledge appears to be only the tip of the possibility is that an additional factor(s) is required for iceberg, however, and many important and fundamental stimulating cohesin’s ATPase and for reconstituting its questions remain to be answered. First, for historical rea-

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Hirano sons, the mitotic function of the SMC protein complexes Adams, R.R., Eckley, D.M., Vagnarelli, P., Wheatley, S.P., Ger- hasbeen emphasizedthusfar. Their interphasefunc- loff, D.L., Mackay, A.M., Svingen, P.A., Kaufmann, S.H., and tionsin recombination and gene regulation need to be Earnshaw, W.C. 2001a. Human INCENP colocalizes with explored more rigorously and more systematically. Sec- the aurora-B/AIRK2 kinase on chromosomes and is overex- ond, our understanding of the meiotic functions of the pressed in tumor cells. Chromosoma 110: 65–74. Adams, R.R., Maiato, H., Earnshaw, W.C., and Carmena, M. SMC protein complexesisfar from complete. For in- 2001b. Essential roles of Drosophila inner centromere pro- stance, surprisingly little is known about the potential tein (INCENP) and aurora B in histone H3 phosphorylation, role of condensin in meiotic chromosome morphogen- metaphase chromosome alignment, kinetochore disjunc- esis. Third, there remains a huge gap in our understand- tion, and chromosome segregation. J. Cell Biol. 153: 865– ing of the bacterial and eukaryotic chromosome cycles. 880. Information from the simple model systems will con- Alexandru, G., Uhlmann, F., Mechtler, K., Poupart, M.-A., and tinuously provide vital hints to the more sophisticated Nasmyth, K. 2001. Phosphorylation of the cohesin subunit actionsof eukaryotic SMC protein complexes.Fourth Scc1 by Polo/Cdc5 kinase regulates sister chromatid cohe- and finally, despite the accumulating information on sion in yeast. Cell 105: 459–472. their cellular functions, we are only beginning to under- Anderson, D.E., Trujillo, K.M., Sung, P., and Erickson, H.P. 2001. Structure of the Rad50/Mre11 DNA repair complex standthe mechanicsof thisunique classoftwo-armed from Saccharomyces cerevisiae by electron microscopy. J. ATPases. Although SMC proteins were originally pre- Biol. Chem. 276: 37027–37033. dicted to be chromatin motors, it is now clear that they Bai, X., Peirson, B.N., Dong, F., Xue, C., and Makaroff, C.A. represent a completely novel type of protein machine. 1999. Isolation and characterization of SYN1,aRAD21-like Future work should integrate knowledge from different gene essential for meiosis in Arabidopsis. Plant Cell approaches including genetics, cell biology, biochemis- 11: 417–430. try, structural biology, and biophysics, and thereby help Bernard, P., Maure, J.-F., Partridge, J.F., Genier, S., Javerzat, J.-P., unveil the highly dynamic nature of chromosome struc- and Allshire, R.C. 2001. Requirement of heterochromatin ture and function. SMC proteins indeed possess the se- for cohesion at centromeres. Science 294: 2539–2542. cret of thisfundamental problem becausethey alwayslie Bhat, M.A., Philp, A.V., Glover, D.M., and Bellen, H.J. 1996. Chromatid segregation at anaphase requires the barren prod- at the heart of the chromosomes. uct, a novel chromosome associated protein that interacts with topoisomerase II. Cell 87: 1103–1114. Bhatt, A.M., Lister, C., Page, T., Fransz, P., Findlay, K., Jones, Acknowledgments G.H., Dickinson, H.G., and Dean, C. 1999. The DIF1 gene of We thank membersof the Hirano laboratory for critically read- Arabidopsis is required for meiotic chromosome segregation ing the manuscript. The work from the author’s laboratory was and belongsto the REC8/RAD21 cohesin gene family. Plant supported by grants from the National Institutes of Health, the J. 19: 463–472. Pew ScholarsProgram in the Biomedical Sciences,and the Hu- Blat, Y. and Kleckner, N. 1999. bind to preferential man Frontier Science Program. sites along yeast chromosome III, with differential regula- tion along armsversusthecentric region. Cell 98: 249–259. Britton, R.A. and Grossman, A.D. 1999. Synthetic lethal phe- notypescausedby mutationsaffecting chromosomeparti- Note added in proof tioning in Bacillus subtilis. J. Bacteriol. 181: 5860–5864. A recent study in S. pombe revealsa direct interaction between Britton, R.A., Lin, D.C.-H., and Grossman, A.D. 1998. Charac- Swi6 and the cohesin subunit Psc3, thereby shedding further terization of a prokaryotic SMC protein involved in chromo- lights on the mechanism by which a subpopulation of cohesin is some partitioning. Genes & Dev. 12: 1254–1259. specifically recruited to pericentromeric heterochromatin Buonomo, S.B.C., Clyne, R.K., Fuchs, J., Loidl, J., Uhlmann, F., (Nonaka, N., Kitajima, T., Yokobayashi, S., Xiao, G., Yama- and Nasmyth, K. 2000. Disjunction of homologous chromo- moto, M., Grewal, S.I.S., and Watanabe, Y. 2002. Recruitment somesinmeiosisIdependson proteolytic cleavage of the of cohesin to heterochromatic regions by Swi6/HP1 in fission meiotic cohesin Rec8 by separin. Cell 103: 387–398. yeast. Nat. Cell Biol. 4: 89–93). Another study by electron mi- Cabello, O.A., Eliseeva, E., He, W., Youssoufian, H., Plon, S.E., croscopy shows that condensin and cohesin display remarkably Brinkley, B.R., and Belmont, J.W. 2001. Cell cycle-dependent different arm conformations, supporting the idea that the two expression and nucleolar localization of hCAP-H. Mol. Biol. SMC protein complexesare structurallydifferentiated to medi- Cell 12: 3527–3537. ate their specialized biochemical and cellular functions (Ander- Chang, G. and Roth, C.B. 2001. Structure of MsbA from E. coli: son, D.E., Losada, A., Erickson, H.P., and Hirano, T. 2002. Con- A homolog of the multidrug resistance ATP binding cassette densin and cohesin display different arm conformations with (ABC) transporters. Science 293: 1793–1800. characteristic hinge angles. J. Cell Biol., in press). Ciosk, R., Shirayama, M., Shevchenko, A., Tanaka, T., Toth, A., Shevchenko, A., and Nasmyth, K. 2000. Cohesin’s binding to chromosomes depends on a separate complex consisting References of Scc2 and Scc4 proteins. Mol. Cell 5: 243–254. Cobbe, N. and Heck, M.M. 2000. SMCsin the world of chro- Adams, R.R., Wheatley, S.P., Gouldsworthy, A.M., Kandels- mosome biology: From prokaryotes to higher eukaryotes. J. Lewis, S.E., Carmena, M., Smythe, C., Gerloff, D.L., and Struct. Biol. 129: 123–143. Earnshaw, W.C. 2000. INCENP binds the aurora-related ki- Collas, P., Le Guellec, K., and Tasken, K. 1999. The A-kinase nase AIRK2 and is required to target it to chromosomes, the anchoring protein AKAP95 isa multivalent protein with a central spindle and cleavage furrow. Curr. Biol. 10: 1075– key role in chromatin condensation at mitosis. J. Cell Biol. 1078. 147: 1167–1179.

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The ABCs of SMC proteins: two-armed ATPases for chromosome condensation, cohesion, and repair

Tatsuya Hirano

Genes Dev. 2002, 16: Access the most recent version at doi:10.1101/gad.955102

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