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Journal of Structural Biology 129, 123–143 (2000)doi:10.1006/jsbi.2000.4255, available online at http://www.idealibrary.com on

Review: SMCs in the World of Chromosome Biology—From Prokaryotes to Higher Eukaryotes

Neville Cobbe and Margarete M. S. Heck1

Institute of Cell and Molecular Biology, University of Edinburgh, Michael Swann Building, King’s Buildings,Mayfield Road, Edinburgh EH9 3JR, United Kingdom

Received December 16, 1999, and in revised form February 25, 2000

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The study of higher order chromosome structurend how it is modified through the course of the cellycle has fascinated geneticists, biochemists, andell biologists for decades. The results from manyiverse technical avenues have converged in theiscovery of a large superfamily of chromosome-ssociated proteins known as SMCs, for structuralaintenance of chromosomes, which are predicted

o have ATPase activity. Now found in all eukaryotesxamined, and numerous prokaryotes as well, SMCslay crucial roles in chromatid cohesion, chromo-ome condensation, sex chromosome dosage compen-ation, and DNA recombination repair. In eukary-tes, SMCs exist in five subfamilies, which appear tossociate with one another in particular pairs toerform their specific functions. In this review, weummarize current progress examining the roleshese proteins, and the complexes they form, play inhromosome metabolism. We also present a twist inhe SMC story, with the possibility of one SMC moon-ighting in an unpredicted location. r 2000 Academic Press

INTRODUCTION

Stretched end-to-end, the DNA in any one cell of auman body would measure about 2 m. Not onlyoes the cell manage to fit this huge length of DNAnto its approximately 5-µm-diameter nucleus, itlso condenses it even further prior to cell division,o that the length of a single DNA molecule isompacted nearly 10,000-fold in the metaphase chro-osome. Two mechanistically distinct but interre-

ated processes are involved in the formation ofitotic chromosomes. In concert with replication or

hortly thereafter, cohesion must be establishedetween sister chromatids and properly maintained

1To whom correspondence should be addressed. Fax: 144 (0)

k31 650 7027. E-mail: [email protected].

123

ntil the metaphase to anaphase transition. Addition-lly, the chromatin must be compacted to yield twoondensed sister chromatids tightly paired at theentromeric regions and also along the length of therms. It is critical for this condensation to happen inn orderly fashion so as to prevent any possiblentanglement or breakage of sister chromatids dur-ng anaphase which would have dire consequences tohe cell. This folding of interphase chromatin to giveaired metaphase chromatids is surely one of theost visually dramatic events of the cell cycle and

ltimately fundamental for ensuring the faithfulegregation of genetic information during cell divi-ion (reviewed in Heck, 1997; Koshland and Strunni-ov, 1996; Murray, 1998).Data suggesting biochemical differences between

nterphase and mitotic chromatin came initiallyrom studies of the synchronized nuclear cycles ofhysarum polycephalum, a true slime mold. Histone1 was found to be extensively hyperphosphorylated

n mitosis (Bradbury et al., 1974; Mueller et al.,985) and strikingly, histone phosphokinase activitydded exogenously to segments of Physarum plasmo-ia was able to accelerate the initiation of mitosisBradbury et al., 1974). Hyperphosphorylation of H1as also observed in CHO cells, and in addition,itotic-specific phosphorylation of serine 10 on his-

one H3 was noted (Gurley et al., 1975). Antibodiesecognizing this highly conserved epitope specificallyabel mitotic chromosomes in all higher eukaryotesxamined to date (Van Hooser et al., 1998; Wei andllis, 1998). Mutation of this particular serine tolanine in Tetrahymena leads to a disruption ofhromosome condensation in mitosis and meiosisWei et al., 1999). These studies point strongly to aole for specific histone H1 and H3 phosphorylationn mitotic chromosome condensation in higher eu-

aryotes.

1047-8477/00 $35.00Copyright r 2000 by Academic Press

All rights of reproduction in any form reserved.

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124 REVIEW: COBBE AND HECK

Not unexpectedly, nonhistone chromosomal pro-eins also play a role in the dramatic reorganizationf higher order chromosome structure during the cellycle. Striking electron microscope images of histone-epleted mitotic chromosomes highlighted the exis-ence of a proteinaceous substructure constraining aea of DNAloops of 50–100 kb (Paulson and Laemmli,977). This ‘‘scaffold’’ fraction, remaining after his-one extraction, was remarkably simple in composi-ion: two major proteins (Sc1 at 170 kDa and Sc2 at35 kDa) and a number of smaller, less abundantroteins (Lewis and Laemmli, 1982). That this frac-ion represented more than an artifact of biochemi-al extraction became clear with the identification ofc1 as topoisomerase II (Earnshaw et al., 1985;asser et al., 1986) and Sc2 as an SMC protein

Saitoh and Laemmli, 1994). Topoisomerase II washown to be essential for chromosome segregation ineast (Holm et al., 1985; Uemura et al., 1987) and, asxpected for a function in chromosome dynamicsuring cell division, was observed to be a marker forroliferating, and not quiescent, cells (Heck andarnshaw, 1986).Additional substantive breakthroughs in our un-

erstanding of chromosome behavior during the cellycle came from the discovery of the SMC family, aovel family of chromosome-associated ATPaseshich appear to have essential and specific roles in

he higher order dynamics of chromosome cohesionnd condensation. The SMC (structural mainte-ance of chromosomes, formerly stability of minichro-osomes) proteins were initially identified through

enetic studies of chromosome segregation in Saccha-omyces cerevisiae (Strunnikov et al., 1993). The firstuch molecule, Smc1p, was originally characterizedy frequent minichromosome nondisjunction in mu-ants (Larionov et al., 1985) and was later shown toe essential for viability and maintaining cohesionetween sister chromatids (Strunnikov et al., 1993).equence comparisons revealed this molecule to be aember of a highly conserved and ubiquitous family.

ndeed, today we know of several structurally dis-inct SMC subgroups playing a key role in chromo-ome dynamics in a host of eukaryotic organisms asell as archaebacteria and many eubacteria (Hi-

ano, 1998, 1999; Jessberger et al., 1998; Koshlandnd Strunnikov, 1996; Strunnikov, 1998; Strunnikovnd Jessberger, 1999). Although no canonical SMCamily members have been found in gram-negativeacteria, similar phenotypes are displayed by Esch-richia coli mutants affecting mukB (Niki et al.,992), which encodes an SMC-like protein (despiteifferences at the termini) with orthologues in otheracteria. Thus, it appears that SMC proteins haven ancient origin, reflecting their fundamental role

n chromosome dynamics. The current phylogeny of d

MC subfamilies and their members is displayed inig. 1.A typical SMC molecule ranges in mass from 115

o 165 kDa and contains five major domains, asnferred from motifs in the amino acid sequence, inhich the N- and C- termini are separated by two

ong coiled-coils of 200–450 residues and a central,lobular hinge region (Jessberger et al., 1998; Peter-on, 1994). The most characteristic motif is the-terminal ‘‘DA’’ box which was noted to have aandidate Walker B motif (ATP hydrolysis signa-ure) (Saitoh et al., 1994; Walker et al., 1982). As the-terminal end of the molecule also contains autative Walker A motif (ATP binding domain), itas suggested that a functional ATPase domain may

orm by uniting the DA box with the ATP-bindingotif (Saitoh et al., 1994). This could occur in either

f two ways: the molecule could bend at the hinge toring the two termini together or by dimerizing as anntiparallel coiled-coil, bringing the N-terminal do-ain of one subunit next to the C-terminal domain of

he other. Indeed, in the case of MukB from E. colind the Smc protein from Bacillus subtilis, it haseen shown that both structures are possible (Melbyt al., 1998). When rotary-shadowed samples of theurified proteins were viewed by electron microscopyEM), they both showed a striking symmetry, appear-ng as a flexible hinge connecting two thin, rod-likerms with terminal globular domains. A range ofifferent conformations were also observed, in whichhe two arms folded tightly against each other orpened up to 180° (separating the terminal globularomains by 100 nm). To distinguish which ends ofhe protein corresponded to the observed globularomains, a modified MukB was created by deletinghe C-terminal domain and replacing the N-terminalomain with a rod-shaped 40-kDa fragment of fibro-ectin. When viewed by EM, the fibronectin domainppeared at both ends, indicating that each half ofhe V-shaped dimer was an antiparallel coiled-coil.lthough the structure of other SMC proteins inifferent organisms has yet to be determined, iteems likely that they share the structure of Smc in. subtilis as a similar basic head–rod–tail structure

s also conserved in even more distantly relatedolecules such as the SbcCD nuclease of E. coli

Connelly et al., 1998). Furthermore, the frictionalatio for the one eukaryotic SMC heterodimer wasound to be similar to that of MukB, suggesting thathe XCAP-C/XCAP-E heterodimer (and possibly otherMC molecules) may adopt a similar conformation.Does the antiparallel dimerization of SMCs gener-

te a functional ATPase? Using the analogue azido-TP, which covalently bonds proteins after lightctivation (Knight and McEntee, 1985), it has been

emonstrated that only the N-terminal domain of

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125REVIEW: STRUCTURAL MAINTENANCE OF CHROMOSOMES

east and mammalian SMCs can directly bind ATPAkhmedov et al., 1998). By contrast, the C-terminalomain appears to be sufficient for DNA bindingAkhmedov et al., 1998; Graumann et al., 1998).owever, the presence of double-stranded DNA haseen shown to stimulate ATP hydrolysis in twoMC-containing complexes (Jessberger et al., 1996b;imura and Hirano, 1997), whereas the B. subtilismc homodimer (which binds preferentially to single-tranded DNA) has a single-strand DNA-stimulatedTPase activity (Hirano and Hirano, 1998). This at

east suggests that ATP hydrolysis might be en-anced by bringing together the respective ATP andNA binding motifs of the termini. Conversely,lthough ATP is not required for DNA binding (Hi-ano and Hirano, 1998; Kimura and Hirano, 1997;imura et al., 1999), it is clearly required for prefer-ntial binding to positively supercoiled substratesKimura et al., 1999). Likewise, ATP binding (thoughot hydrolysis) is also required for the enhancedggregation of the B. subtilis Smc with ssDNAHirano and Hirano, 1998). Of course, it remains toe seen if ATP hydrolysis itself is strictly abolishedy removing the C-terminal domain. However, thebility to form antiparallel dimers is not necessarilyufficient to generate a functional ATPase. For ex-mple, the ATP-stimulated activity of a XenopusMC complex involved in chromosome condensationepends on the presence of additional non-SMCubunits (Kimura et al., 1998, 1999) and the SMCeterodimer of the RC-1 recombination compleximilarly requires other components for full ATPasectivity (Jessberger et al., 1996b). In any event, ATPydrolysis appears to be required for the full func-ion of SMC-containing complexes, as shown byutagenesis of the ATP-binding domain (Chuang et

l., 1994; Verkade et al., 1999) or the use of nonhydro-yzable ATP analogues (Kimura and Hirano, 1997).

The eukaryotic SMCs may be divided into fiveajor groups, including the Rad18 subfamily and

our other subfamilies whose members may combines heterodimers in larger functional complexes. Whilehe Rad18 members are only essential for DNAepair (Lehmann et al., 1995; Mengiste et al., 1999;erkade et al., 1999), the other SMC moleculesppear to have essential nonoverlapping functions,s examples of each type are known to be requiredor viability (Holt and May, 1996; Michaelis et al.,997; Saka et al., 1994; Strunnikov et al., 1993,995). The ability of particular eukaryotic SMColecules to combine as heterodimers is suggested

y their co-immunoprecipitation in roughly equimolarmounts (Darwiche et al., 1999; Hirano and Mitchi-on, 1994; Lieb et al., 1998; Losada et al., 1998;chmiesing et al., 1998; Sutani et al., 1999) and by

nalogy with the observed homodimerization of bac- (

erial SMCs (Melby et al., 1998). Although the poten-ial for eukaryotic SMCs to form homodimers haslso been demonstrated, this nonpreferential associa-ion only occurred when the fusion proteins wereighly overexpressed, thereby titrating out the natu-al SMC partner (Strunnikov et al., 1995). Moreover,t seems that homodimerization is insufficient for

ost eukaryotic SMCs to function as their in vitroctivity depends on the combined presence of bothubunits (Kimura and Hirano, 1997; Kimura et al.,999; Schmiesing et al., 1998; Sutani and Yanagida,997) and mutation of just one SMC partner pro-uces defects in vivo (Chuang et al., 1994; Lieb et al.,998; Michaelis et al., 1997; Saka et al., 1994;trunnikov et al., 1993, 1995). On the other hand,igher levels of oligomerization would appear to berecluded by considering the overall mass of isolatedMC-containing complexes and the known mass ofhe other components (Hirano et al., 1997; Hiranond Mitchison, 1994; Losada et al., 1998; Sutani andanagida, 1997; Sutani et al., 1999). So far, twoundamental classes of SMC heterodimer have beenescribed in various organisms (Heck, 1997; Hirano,999; Hirano et al., 1995; Jessberger et al., 1998).hese heterodimers may associate with differentets of non-SMC subunits to yield a range of largerotein complexes with diverse functions, as shownn Tables I and II and Fig. 2. The SMC2/SMC4eterodimer seems to have a role in mediatingitotic chromosome condensation, as part of the

‘condensin complex’’ (Hirano and Mitchison, 1994;utani and Yanagida, 1997; Sutani et al., 1999). AnMC2 homologue and another SMC4-like moleculere implicated in sex chromosome dosage compensa-ion in Caenorhabditis elegans (Chuang et al., 1994;ieb et al., 1996, 1998). On the other hand, theMC1/SMC3 heterodimer forms part of a complex

mportant for sister chromatid cohesion, dubbed‘cohesins’’ (Guacci et al., 1997; Losada et al., 1998;

ichaelis et al., 1997; Tóth et al., 1999), and is alsonvolved in recombination as part of the RC-1 com-lex (Jessberger et al., 1996a,b).Unlike the SMC proteins of eukaryotes, no cofac-

ors for the B. subtilus Smc have been isolated toate (Sharpe and Errington, 1999). Nonetheless, aole for the protein in chromosome structure andartitioning was clearly demonstrated by the abnor-al nucleoids and accumulation of anucleate cells in

mc mutants (Britton et al., 1998; Moriya et al.,998). Similar phenotypes were also observed in smcull mutants of Caulobacter crescentus (Jensen andhapiro, 1999). The B. subtilus smc mutants werelso characterized by irregular subcellular localiza-ion of Spo0J [a chromosome partitioning proteinhich binds to sites near the origin of replication

Lin and Grossman, 1998)]. The role of the Smc

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FIG. 1. Phylogenetic tree of known SMC proteins. The aboequences generated by the ClustalW program (Methods Enzymolrees produced by the ClustalW program were checked by resamplnferred using the PROTDIST and NEIGHBOR programs of Joe Fhylip.html). The topology of the branches comprising each indivisconsin Package to construct separate alignments and then u

AUP program (http://www.lms.si.edu/PAUP/about.html) to find tf subfamilies were confirmed by analysis of distance matrix datubfamily was compared with the mean distance of each subfamilree shown was confirmed by ClustalW alignment of partial dataultiple substitutions, in addition to comparison with trees base

orrect topology of the more distantly related SMC-like proteinsestFit program in the Wisconsin Package and then constructing

length of shorter protein 4 length of alignment) 3 (100% 2 % idend ClustalW programs were also compared with branch lengths oHYLIP and ClustalW trees were viewed using Rod Page’s TreeVi

ve tree was constructed based on alignments between SMC protein. 266, 383–402, 1996), correcting for multiple substitutions. The variousing with 1000 bootstrap trials and compared with neighbor-joining treeselsenstein’s PHYLIP package (http://evolution.genetics.washington.edu/idual subfamily was also confirmed by using the PileUp program in thesing a GCG interface to the tree-searching options of David Swofford’she optimal topology by means of parsimony. Moreover, the designationsa, in which the mean distance between all members of the same SMCy member from all other proteins in the tree. The overall topology of thesets of full-length SMC molecules both with and without correction for

d on alignment of the conserved N- and C-terminal domains alone. Thewas confirmed by conducting all possible optimal alignments using thea neighbor-joining tree from pairwise distances calculated as distance 5ntity). The branch lengths in the overall tree calculated by the PHYLIPf trees containing only members of the same SMC subfamily. Finally, theew program (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html).

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rotein in B. subtilus is reflected by its chromosomalocalization and its presence at the poles of theucleoid as discrete foci (Britton et al., 1998; Grau-ann et al., 1998). As the N-terminal region of Smc

s required for the formation of polar foci (Graumannt al., 1998), it appears to be needed for pairing ofewly replicated origins (Lin and Grossman, 1998)y mediating Spo0J localization to the pole of theucleoid and thereby facilitating orderly segrega-ion. However, due to the small size of bacterial cellst remains unclear whether the mutant phenotypesre caused primarily by a defect in chromosome conden-ation, segregation, or both (Sharpe and Errington,999). Nonetheless, an insight into the possible conden-ation activity of the B. subtilis Smc homodimer hasome from the discovery of its ATP-dependent DNAeannealing activity (Hirano and Hirano, 1998). Asacterial nucleoids contain unconstrained negativeupercoils that may be easily unpaired (Pettijohn,

FIG. 2. Model of SMCs, from monomer to heterodimer to highomain near its N-terminus and an ATP hydrolysis motif near iexible hinge region near the center of the molecule. When heterolose proximity to the Walker B motif of its partner SMC. SMCubunits, diagrammed as three ovals, resulting in creation of the c

982), it has been proposed that the energy-depen- a

ent aggregation of single-stranded DNA may com-act bacterial chromosomes by bringing such regionsogether (Hirano and Hirano, 1998). Furthermore,he increase in twist resulting from SMC-mediatedestoration of base pairing may compact the DNAhrough the concomitant generation of compensatoryositive supercoils (Sutani and Yanagida, 1997).As DNA reannealing activities similar to that of

he recombination protein recA (Weinstock et al.,979) have also been observed with the S. pombeut3/cut14 heterodimer (Sutani and Yanagida, 1997),he bovine bSMC1/bSMC3 heterodimer (Jessbergert al., 1996b), and even the isolated C-terminal do-ains of Smc1p and Smc2p from S. cerevisiae (Akh-edov et al., 1998), it has been suggested that thisay represent an activity characteristic of all SMColecules (Yanagida, 1998). However, these eukary-

tic proteins differ from the B. subtilis Smc ho-odimer as they do not require ATP for reannealing

er functional complexes. The SMC monomer has an ATP bindingrminus. The regions are separated by coiled-coil domains and azed in an antiparallel fashion, one Walker A motif is brought intorodimers then participate in complex formation with non-SMCand condensin complexes.

er ordts C tedimeri

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997). As SMCs do not seem to translocate directlylong the DNA (Kimura and Hirano, 1997), the rolef molecules such as bSMC1 and bSMC3 in recombi-ation may be related to their role in cohesion, inhich recombination is facilitated by keeping chro-

TAProtein Complexes Co

Species

SMC subunits

SMC2 type SMC4 type

ondensin complexS. cerevisiae Smc2 (Strunnikov et

al., 1995)Smc4

S. pombe Cut14 Cut3

C. elegans MIX-1 Z69787

D. melanogaster dmSMC2 dmSMC4

X. laevis XCAP-E (Hirano andMitchison, 1994)

XCAP-C (Hirano aMitchison, 1994

G. gallus ScII (Saitoh et al.,1994)

?

H. sapiens hCAP-E (Schmiesinget al., 1998)

hCAP-C (Schmieset al., 1998)

osage compensationC. elegans MIX-1 (Lieb et al.,

1998)DPY-27 (Chuang e

1994, 1996)

TAProtein Complexes Co

Species

SMC subunits

SMC1 type SMC3 type

ohesin complexS. cerevisiae Smc1 (Strunnikov et

al., 1993)Smc3 (Michaelis et al

1997)

S. pombe CAA22432 CAA15722

A. nidulans ? SudA (Holt and May,1996)

C. elegans AAB93638 CAB57898

D. melanogaster dmSMC1 dCAP (Hong andGanetsky, 1996)

X. laevis XSMC1 (Losadaet al., 1998)

XSMC3 (Losada et al1998)

M. musculus SMCB (Darwiche etal., 1999)

SMCD (Darwiche etal., 1999)

R. norvegicus SMC1 Bamacan (Wu andCouchman, 1997)

H. sapiens hSMC1 (Rocques etal., 1995; Schmi-esing et al., 1998)

hCAP (Shimizu et al.1998)

ecombination com-plex

B. taurus bSMC1 (Jessberger etal., 1996)

bSMC3 (Jessberger eal., 1996)

atids close together. However, this does not explainhe renaturation activity of SMCs that are active inhromosome condensation but not cohesion. More-ver, as the isolated C-terminal domains of Smc1pnd Smc2p are capable of efficient DNA reannealing

Iing SMC2 and SMC4

Non-SMC subunits

AAB67384 Brrn1 CAB41223

cnd1 (Sutani et al.,1999)



CAA16340 ? ?

EST clot No. 2519 Barren (Bhat et al.,1996)

EST clot No. 2199

XCAP-D2 (Kimura etal., 1998)

XCAP-H (Hirano etal., 1997)

XCAP-G (Hirano etal., 1997)

? ? ?

063880 038553 ?

DPY-28 (Lieb et al.,1998)

DPY-26 (Lieb et al.,1996)

?

IIing SMC1 and SMC3

Non-SMC subunits

1/Scc1 (Guacci et., 1997; Michaelisal., 1997)

Scc2 (Michaelis et al.,1997)

Scc3 (Tóth et al., 1999)

21 (Birkenbihld Subramani,92)

Mis4 (Furuya et al.,1998)

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? ? ?

09926 Nipped-B (Rollins etal., 1999)

Stromalin (Valdeolmil-los et al., 1998)

D21 (Losada et., 1998)

p155 (Losada et al.,1998)

p95 (Losada et al.,1998)

9 (Darwiche et., 1999)

? Stromal antigen 1 (Car-romolino et al., 1997)

? ? ?

D21 (McKay et., 1996)

? Stromal antigen 1 (Car-romolino et al., 1997)

ligase III (Jess-rger et al., 1993)

DNA Pol e (Jessbergeret al., 1996)

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n their own,2 it seems that the ability of SMCs toether separate DNA molecules may simply enhancehe reannealing activity by helping to bring ssDNAogether. Clearly, more detailed analysis of the reac-ions catalyzed by the terminal domains is requiredo determine the mechanism of SMC-mediated recom-ination. Lastly, as SMC molecules have a higherffinity for AT-rich sequences such as SARs andARs (Scaffold- or Matrix- Associated Regions)

Akhmedov et al., 1998) which may be more easilyelted (Bode et al., 1992), it is possible that the

enaturing activity of different SMC protein com-lexes in eukaryotes might be linked to roles inondensation and segregation, as in B. subtilus. Weill now look more closely at the role of SMCs in

hese two process, beginning with their involvementn chromosome condensation.

SMCs AND CHROMOSOME CONDENSATION

The first SMC proteins exhibiting a role in chromo-ome condensation were found in S. pombe. The cut3nd cut14 mutants displayed a characteristic ‘‘cut’’cell untimely torn) phenotype, in which the divisioneptum bisects the nuclear material, due to a failuren either chromosome condensation or sister chroma-id segregation (Saka et al., 1994). However, it wasuggested that the primary defect was a failure inhromosome condensation as high rates of minichro-osome loss were not observed in cut3 mutants and

entromeric DNA was reported to segregate properlyo the spindle poles in both cut3 and cut14 mutants.he improper chromosome disjunction in these mu-ants therefore appeared to be a consequence ofmpaired chromosome condensation. Similar pheno-ypes were also observed in S. cerevisiae smc2 mu-ants, encoding a Cut14p orthologue (Strunnikov etl., 1995). Cut3p was later shown to be orthologouso Smc4p in S. cerevisiae (Koshland and Strunnikov,996). In addition, the Sc2 protein of the mitotichromosome scaffold in chicken cells was identifieds an SMC2 subfamily member, suggesting a pos-

2Considering that similar molar concentrations were used forhe reactions with either full-length or partial SMC proteins, theeannealing activity of isolated SMC terminal domains appears toonflict with the inability of full-length SMCs to promote duplexormation unless they can heterodimerize (Hirano, 1999). Fortu-ately, this discrepancy may be explained by considering theollision rates of the different proteins with DNA. Presumably, themall, globular truncated protein has a higher collision rate thanhe long, rod-shaped full-length molecule because it is able toiffuse through solution more easily. Likewise, a heterodimerormed from two different SMCs has a COOH-terminal DNAinding domain at each end so it is more likely to bind to DNA. Byontrast, as individual SMC proteins are unlikely to form ho-odimers unless they are produced in vast excess (Strunnikov et

l., 1995), they will only be able to bind DNA at one end and so will

Cave a far lower collision rate.

ible structural role for these proteins in mitotichromosome architecture (Saitoh et al., 1994).Much of our current understanding of chromo-

ome condensation is based on the in vitro simula-ion of chromosome condensation when nuclei aredded to mitotic extracts from Xenopus eggs. Topo-somerase II was shown to be required for mitotichromosome condensation when either HeLa orhicken erythrocyte nuclei or demembranated spermere added to Xenopus extracts (Adachi et al., 1991;irano and Mitchison, 1993). A heterodimeric com-lex containing XCAP-E (SMC2-type) and XCAP-CSMC4-type) was identified as a mitotic chromo-omal component in in vitro assembled chromosomesusing demembranated sperm as substrate), andmmunofluorescence detection of XCAP-C revealed alamentous distribution along the chromosome axis

Hirano and Mitchison, 1994), not unlike that ob-erved for topoisomerase II in ‘‘normal’’ mitotic chro-osomes (Earnshaw et al., 1985; Earnshaw andeck, 1985). Two different types of condensin com-lex were later identified by sucrose gradient sedi-entation, namely an 8S form which proved to be

he XCAP-E/XCAP-C heterodimer and a larger 13Somplex containing three additional subunits (re-erred to as XCAP-D2, XCAP-H, and XCAP-G) (Hi-ano et al., 1997). Both the targeting of the condensinomplex to chromosomes and its in vitro activityere shown to depend on mitosis-specific phosphory-

ation of these additional non-SMC subunits (Hiranot al., 1997), with p34cdc2 responsible for the hyper-hosphorylation of XCAP-D2 and XCAP-H (Kimurat al., 1998). XCAP-H was also found to have homol-gy to Barren, a protein localizing to the chromo-omes of mitotically active cells in Drosophila em-ryos (Bhat et al., 1996). Mutants in barren wereharacterized by extensive chromatin bridges be-ween anaphase chromosomes, in spite of centro-ere separation. This phenotype was reminiscent of

he cut3 and cut14 mutants in S. pombe, consistentith a role for Barren in proper mitotic chromosome

ondensation.A larger condensin complex has also been detected

n S. pombe cell lysates, with a similar subunitomposition to that of the Xenopus 13S condensinomplex (Sutani et al., 1999). Gene disruption hasemonstrated that the additional non-SMC subunitsre essential for viability and the mutants wereharacterized by hypocondensed chromosomes whichere extended along an elongated spindle instead of

learly separating, as seen in cut3 and cut14 cells.hereas the activity of the Xenopus condensin com-

lex is regulated by phosphorylation of its non-SMCubunits, that of the S. pombe condensin appears toe controlled by mitosis-specific phosphorylation of

cdc2

ut3p by p34 . This modification was also shown

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o be essential for viability as it restricts the accessi-ility of a nuclear export signal (NES) in the N-erminus of the protein. As the intracellular shut-ling of the other condensin subunits seems to dependn the localization of Cut3p, the phosphorylation of

FIG. 3. Model for cohesin and condensin deposition and activhromatin either coincident with or shortly after DNA replicationnaphase. The condensin complex (blue circles) then appears to bee thought of as a two-step process resulting first in shortening ofhe final high degree of chromosome compaction. Cohesion betwissolved at the transition to anaphase, and the sisters are segreuclear envelope reformation and cytokinesis, the chromatin is de

ut3p during mitosis permits entry of the complex M

nto the nucleus while the dephosphorylated formxposes the NES during interphase and thereforeelegates the complex to the cytoplasm. This mannerf regulating condensin activity contrasts with thatf the Xenopus condensin complex (Hirano and

ing the cell cycle. The cohesin complex (red ovals) is loaded ontohase, thereby ensuring the attachment of sister chromatids untilduring chromosome condensation in prophase. Condensation canterloop axis, followed by the introduction of supercoils to achievee sister chromatids of the final metaphase chromosome is thenby the microtubule apparatus to the poles of the cell. Followingsed in preparation for transcription and DNA synthesis.

ity durin S ploadedthe in

een thgated

conden

itchison, 1994) and the chicken Sc2 protein (Saitoh

edctdsatsdbscnh

aanrcabimSracctbaas(osepiH

stecD1acf(i

s(wht‘(tttp1hbicwciw

ftdhotipacptmlsflp(nlDotmtcpwhptpo


t al., 1994), which both remain in the nucleusuring interphase but fail to associate with thehromosomes until mitosis. Until it can be shownhat the essential activity of the S. pombe complexepends on the phosphorylation of any of its otherubunits, one possible explanation for this shuttlingppears to be that the complex is active throughouthe cell cycle. Consequently, it appears that chromo-ome condensation during mitosis may be regulatedifferently in different species although the sameasic protein complex appears to be involved. Onehould also keep in mind that S. pombe undergoeslosed nuclear mitosis, in contrast to the observeduclear envelope breakdown of Xenopus and otherigher eukaryotes.Purified Cut3p and Cut14p form a stable complex

t a rather low efficiency when mixed in vitro (Sutanind Yanagida, 1997), so it has been proposed that theon-SMC subunits Cnd1p and Cnd3p might have aole in linking the SMCs together in the functionalondensin complex. This is suggested by the report ofweak homology between Cnd1p and Cnd3p and thesubunit of the AP3 adapter protein complex, which

s involved in the assembly of rod-like clathrinolecules for vesicle transport (Sutani et al., 1999).ince both clathrin and SMC molecules contain twood-like regions linked by a hinge-like region, Cnd1pnd Cnd3p may enable the rod-like SMC subunits toorrectly assemble. The intact Xenopus condensinomplex was also shown to preferentially bind posi-ively supercoiled DNA and DNA with a distorted orent structure, such as a four-way junction (Kimurand Hirano, 1997; Kimura et al., 1999). A similarffinity for cruciform DNA has also been demon-trated with Smc1p and Smc2p from S. cerevisiaeAkhmedov et al., 1998). As bending and supercoilingf the DNA may be generated by the SMCs, thisuggests that additional condensins might bind coop-ratively (Kimura and Hirano, 1997), just as otherroteins which strongly bind cruciform or bent DNAn vitro tend to bend it further (Zlatanova and vanolde, 1998).

MECHANISM OF CONDENSIN ACTION

How is the condensin complex involved in chromo-ome condensation? Although renaturation can con-ribute to supercoiling, this activity fails to fullyxplain the role of certain SMCs in chromosomeondensation. After all, the ability to promote duplexNA is shared by other SMCs (Jessberger et al.,996b) and indeed non-SMC proteins (Weinstock etl., 1979) which have no obvious direct role inondensation. As the reannealing reaction is there-ore considered to be only a part of its activityYanagida, 1998), how does the condensin complex

nteract with chromatin to induce its mitotic conden- c

ation? Based on the symmetrical structure of BsSMCMelby et al., 1998) in which each end could interactith both ATP and DNA (Akhmedov et al., 1998), itas been suggested that the homodimer may func-ion as an ATP-modulated DNA cross-linker with a‘scissoring’’ action to induce aggregation of DNAHirano, 1999). The possibility of such conforma-ional changes during SMC activity is supported byhe finding that sensitivity to proteolytic cleavage ofhe B. subtilus SMC homodimer depends on theresence of ATP and ssDNA (Hirano and Hirano,998). By extending this concept to eukaryotic SMCeterodimers, this scissoring action was proposed toe the key mechanism underlying all SMC activities,n which the SMC2/SMC4 heterodimers involved inhromosome condensation and dosage compensationould act as intramolecular DNAcross-linkers which

ompact a single DNA molecule (Hirano, 1999). Whatmpact native chromatin, in contrast to naked DNA,ould have on this proposed process is anyone’s guess.An early model for condensin action was put

orward by Kimura and Hirano when they foundhat the Xenopus 13S condensin complex can intro-uce positive supercoils into DNA, fueled by ATPydrolysis (Kimura and Hirano, 1997). The stretchesf DNA between condensin binding sites could formwisted loops by compensatory negative supercoil-ng, which are relaxed by treatment with eitherrokaryotic or eukaryotic type I topoisomerases. Ingreement with previously suggested mechanisms ofompaction based on the chromosome scaffold modelroposed originally by Paulson and Laemmli (1977),he authors proposed that chromosome condensationight be initiated by the formation of chromatin

oops by condensin-mediated supercoiling at specificites, followed by shortening of the interloop axis andolding of the torsionally constrained loops. It wasater shown that condensin reconfigures DNA in theresence of a type II topoisomerase by creating knotsKimura et al., 1999). As knotting presumably wouldot occur if condensin generated supercoils either by

ocally overwinding the DNA or by wrapping theNA around itself, this implied that the complexperated by generating a global writhe. When theopology of the knots was determined by electronicroscopy of RecA-coated DNA, it was reported that

he vast majority were positive, implying that theondensin complex generated an ordered array ofositive solenoidal supercoils. As the condensinsere reported to bind to plasmid DNA in vitro at aigh ratio (Kimura et al., 1998, 1999), it was pro-osed that a high density of condensins could bindhe full DNA length, touching each other to form arotein infrastructure capable of nonplanar bendingf the DNA. This model neatly explains both the

ooperativity of condensin binding and the observed

pHthttsi

swHtvs(crsappsmas(nScndptistpci

cpladpmtpptnciF

tbtefcasccsto1anp1cadb

baCoactevrtt(wbswttaaMmtcDtmm


reference for longer DNA fragments (Kimura andirano, 1997) in terms of cooperative binding. Fur-

hermore, as the two ends of the eukaryotic SMCeterodimer are similar but not identical (containinghe N- and C-termini of different SMC molecules),his asymmetry might contribute to the chirality ofupercoiling, provided that the complex binds DNAn a fixed orientation.

Although the high concentrations of condensinsupplied in vitro allowed the complex to bind every-here on the naked plasmid DNA (Hirano andirano, 1998; Kimura et al., 1999), it is unlikely that

he same is true of protein-laden chromosomes inivo. Otherwise, this would conflict with the ob-erved distribution of condensin SMCs in XenopusHirano and Mitchison, 1994), chicken, and humanells (Saitoh et al., 1994), in which they appearedestricted to the chromosome axis with concentratedtaining at the centromeres (Saitoh et al., 1994). Thebundance of Cut3p in wild-type S. pombe cellsredicted a density of only one condensin complexer 8 kb of DNA (Sutani and Yanagida, 1997) and aimilar stoichiometry was estimated for Xenopusitotic chromosomes assembled in vitro (Kimura et

l., 1999). As SMC proteins were previously ob-erved to preferentially bind AT-rich sequencesAkhmedov et al., 1998), it seems possible thatonplanar bending might be initiated at loci such asARs. By combining these findings, one model forondensation by condensins would result from theonplanar bending of DNA by higher order multicon-ensin complexes, generating positive solenoidal su-ercoils at defined sites along the chromosome andhe simultaneous generation of negative supercoilsn the intervening regions. Although these negativeupercoils could be easily removed by the numerousopoisomerases in a cell, this would presumably berevented by the binding of additional unidentifiedondensation factors which could stabilize thesenterwound loops.

Despite the insights provided by these models ofondensation, the precise mode of action of SMCroteins continues to provoke discussion. In particu-ar, the interaction between condensin complexesnd topoisomerase II still remains enigmatic. Evi-ence of possible genetic interactions was initiallyrovided by analysis of different cut3 and topoIIutants in S. pombe (Saka et al., 1994), although

hese results are equally consistent with the tworoteins acting in a common pathway without directhysical interaction. A functional interaction be-ween topoisomerase II and Barren was suggested,onetheless, based on co-immunoprecipitation, colo-alization on mitotic chromosomes, and interactionn a yeast two-hybrid assay (Bhat et al., 1996).

urthermore, Barren has been reported to enhance i

he supercoiling activity of topoisomerase II, possi-ly modulating topoisomerase II-mediated decatena-ion of chromosomal arms. Finally, Sc2 and topoisom-rase IIa have been reported to copurify in a complexound in undifferentiated mouse erythroleukemiaells (Ma et al., 1993) and the two proteins cofraction-te with and colocalize to the mitotic chromosomecaffold of chicken cells (Saitoh et al., 1994). Inontrast, the Xenopus condensins fail to immunopre-ipitate with topoisomerase II (Hirano and Mitchi-on, 1994) and appear to be independently targetedo mitotic chromosomes (Hirano et al., 1997). More-ver, unlike topoisomerase IIa and b (Berrios et al.,985; Meyer et al., 1997; Petrov et al., 1993; Zini etl., 1992), Sc2 is not a component of the interphaseuclear matrix, as it readily leaked into the cyto-lasm during subcellular fractionation (Saitoh et al.,994). Regardless of whether the members of theondensin complex interact directly with topoisomer-se II, it is clear that their respective condensing andecatenating activities contribute synergistically toring about chromosome condensation.Intriguingly, there may be a functional similarity

etween mechanisms of chromosome condensationnd the global regulation of gene expression on the. elegans X chromosome, based on the involvementf an SMC2/4 heterodimer in sex chromosome dos-ge compensation. Transcription from each of the Xhromosomes is reduced in hermaphrodites (XX) ofhis organism to match the level of X-linked genexpression in males (XO). The discovery that aariant SMC4 type protein (DPY-27) is an essentialegulator of dosage compensation through its associa-ion with the X chromosome provided the first cluehat SMC proteins might be involved in this processChuang et al., 1994, 1996). Subsequently, MIX-1as identified as an SMC2-type protein required foroth mitosis and dosage compensation, the re-tricted localization of which to the X chromosomeas dependent on DPY-27 (Lieb et al., 1998). Like

he 13S condensin complex, the dosage compensa-ion complex consists of an SMC2/4 heterodimer andt least two non-SMC subunits, including DPY-26nd DPY-28 (Hirano, 1999). The mitotic function ofIX-1 is achieved through its association with aore conventional SMC4-type protein, suggesting

hat MIX-1 may have been enlisted to the dosageondensation complex through the evolution ofPY-27 as a highly specialized SMC protein, altering

he higher order structure of X chromosomes by aechanism perhaps related to that underlying chro-osome condensation.

SMCs AND SISTER CHROMATID COHESION

Another aspect of mitotic chromosome dynamics

n which the eukaryotic SMC proteins play a funda-

mssw1fia(fiqMisarfc(epwteSwaactcsttkRt1

dsmsaeacFcasltfaiD

mmstmsatlaqcP1taA(ldfaitmatXalb1

ptfgets(mtseiltTftr

mt


ental role is the establishment and maintenance ofister chromatid cohesion. The first SMC moleculehown to have a vital role in maintaining cohesionas Smc1p (Larionov et al., 1985; Strunnikov et al.,993). Its Smc3p partner was subsequently identi-ed in a genetic screen for S. cerevisiae mutants withpremature sister chromatid separation phenotype

Michaelis et al., 1997). The same screen also identi-ed Scc1p and Scc2p, two non-SMC proteins re-uired for sister chromatid cohesion. Meanwhile,cd1p (identical to Scc1p) was also identified in

ndependent screens for high-copy suppressors of anmc1 mutant or mutants displaying enhanced invi-bility after mitotic arrest (Guacci et al., 1997). Theole of this protein in chromosome segregation wasurther verified by the heightened instability ofircular minichromosomes in an scc1/mcd1 mutantHeo et al., 1998). The functional association ofither Scc1p/Mcd1p or Scc3p (another essential com-onent of the cohesin complex) with chromosomesas then found to depend on each other and also on

he presence of Smc1p, Smc3p, and Scc2p (Michaelist al., 1997; Tóth et al., 1999). In particular, themc1p, Smc3p, Scc1p/Mcd1p, and Scc3p proteinsere found to co-immunoprecipitate in roughly equalmounts, implying that they maintained cohesion ascomplex (Guacci et al., 1997; Tóth et al., 1999),

oined ‘‘cohesin.’’ By contrast, Scc2p does not appearo be a stoichiometric component of the cohesinomplex and fails to colocalize with other cohesinubunits on chromosomes but is nonetheless essen-ial for the binding of these other subunits to chroma-in (Tóth et al., 1999). Its orthologue in S. pombe,nown as Mis4, failed to coprecipitate with eitherad21p (the S. pombe orthologue of Scc1p/Mcd1p) or

he S. pombe orthologue of Smc3p (Furuya et al.,998).The need to establish cohesion during S phase was

emonstrated in cells expressing Scc1p/Mcd1p exclu-ively from a galactose-inducible promoter (Uhl-ann and Nasmyth, 1998). Ctf7p/Eco1p was later

hown to be another S. cerevisiae protein whosectivity is essential to establish cohesion along thentire length of the chromosome during S phase,lthough it was not required for the maintenance ofohesion (Skibbens et al., 1999; Tóth et al., 1999).urthermore, Ctf7p/Eco1p does not seem to be aohesin subunit and its presence has no effect on thessociation of the cohesin complex with chromo-omes (Tóth et al., 1999). Interestingly, a syntheticethal interaction was observed between a tempera-ure-sensitive mutation in ctf7 and the yeast genesor either PCNA or an RFC-like protein (Skibbens etl., 1999), suggesting that PCNA might be involvedn loading the cohesin complex onto chromatin after

NA replication. It has been suggested that PCNA p

ay play a role in the assembly of chromatin (Kel-an, 1997) as its nuclear distribution in fertilized

tarfish eggs coincided with the chromatin distribu-ion during the first S phase (Nomura, 1994) andutations in the Drosophila gene encoding PCNA

uppressed position-effect variegation (Henderson etl., 1994). In addition, PCNA has been shown to bindhe largest subunit of CAF-1, demonstrating a directink between replication machinery and chromatinssembly (Shibahara and Stillman, 1999). Conse-uently, it was proposed that the loading of cohesinomplexes onto chromatin might be coupled withCNA-dependent DNA replication (Skibbens et al.,999). This adds weight to a previous hypothesishat cohesion might be directly coupled to passage of

replication fork (Uhlmann and Nasmyth, 1998).lthough the murine orthologue of Scc1p/Mcd1p

known as PW29) and PCNA fail to exhibit similarocalization patterns (Darwiche et al., 1999), thisoes not necessarily preclude a role for PCNA inacilitating the loading of cohesins onto chromatin,s Scc1p/Mcd1p in S. cerevisiae also does not colocal-ze with Scc2p, even though the latter is essential forhe efficient binding of Scc1p and other cohesinembers to chromatin (Tóth et al., 1999). Similarly,

lthough the binding of cohesin subunits to chroma-in occurred independently of DNA replication inenopus oocyte extracts (Losada et al., 1998), thislso does not prevent a possible role for PCNA as aanding pad for SMC proteins as PCNA may alsoind to DNA at times other than S phase (Nomura,994).The similarities between the S. cerevisiae and S.

ombe proteins involved in regulating sister chroma-id cohesion, combined with the conservation ofactors required for chromosome condensation, sug-ested that the cohesion mechanism might also bevolutionarily conserved. Indeed, the vertebrate or-hologues of SMC1, SMC3, and SCC1 have also beenhown to be essential for sister chromatid cohesionLosada et al., 1998) and proper progression ofetaphase (Schmiesing et al., 1998), even though

hese proteins appear to dissociate from chromo-omes during mitosis (Darwiche et al., 1999; Losadat al., 1998; Schmiesing et al., 1998). Nevertheless,mmunoblotting showed that the murine ortho-ogues of both SMC1 and SMC3 were expressedhroughout the cell cycle (Darwiche et al., 1999).his dissociation of the vertebrate cohesin complex

rom mitotic chromosomes contrasts with the pat-ern observed in yeast, in which Smc1p and Smc3pemain associated (Michaelis et al., 1997).Although it is possible that cohesion at this stageight be supported by other molecules (as yet uniden-

ified), it has been shown that hSMC1 nonetheless

lays a role in the maintenance of chromatid cohe-

spsmmewqtsmtoadatrTmltpe((oicc

chmtbAthS(stMbd

cbcbnDr

1bssiasnd1weKqrn1SD1ictts(sDageafarli

cin1mtqtpcstfpsbt


ion as well as its establishment, even though therotein appears to be excluded from the chromo-omes during mitosis. This was demonstrated by theitotic arrest of HeLa cells microinjected duringid/late metaphase with an antibody specific for

ither the middle or C-terminal regions of hSMC1,hereas cells injected at early anaphase subse-uently went through cytokinesis normally to yieldwo daughter cells (Schmiesing et al., 1998). Thisuggests that a residual level of cohesins bound toetaphase chromosomes may be sufficient to main-

ain cohesion between sister chromatids until thenset of anaphase, as previously proposed (Losada etl., 1998). Moreover, it has been proposed that theissociation of most cohesins from the chromosomest the onset of mitosis in vertebrate cells may loosenhe linkage between sister chromatids, permittingeorganization of the chromatin (Losada et al., 1998).his may serve to relieve a steric barrier whichight otherwise prevent final condensation in such

arge chromosomes, as mediated by replacement ofhe cohesins by the condensins. This idea is sup-orted by the prevention of interphase cells fromntering mitosis by overexpression of an PW29SCC1)–GFP fusion protein in mouse fibroblastsDarwiche et al., 1999). Transfection with H2B–GFPr the GFP molecule itself produced no such arrest,mplying that the SCC1 (PW29) protein and itsomplex with SMC proteins might be involved in theontrol of mitotic cycle progression.So what function do the SMC proteins fulfill in the

ohesin complex? Assuming that the SMC1/SMC3eterodimers of cohesin complexes function as ATP-odulated DNA cross-linkers, it has been suggested

hat these molecules may form intermolecular bridgesetween separate DNA molecules (Hirano, 1999).lternatively, such bridges might be produced

hrough the association of two different Smc1/3eterodimers (possibly mediated by Scc1p/Mcd1p orcc3p), each of which is bound to a single chromatid

Losada et al., 1998). This latter model seems plau-ible for S. cerevisiae, as dissolution of sister chroma-id cohesion can be achieved by cleavage of Scc1p/cd1p (Ciosk et al., 1998; Uhlmann et al., 1999) and

oth Smc1p and Smc3p persist after Scc1p/Mcd1pissociation (Tanaka et al., 1999).

LOCALIZATION OF THE COHESIN COMPLEX

Having established the importance of the cohesinomplex for sister chromatid cohesion, where does itind on the chromosome? The relative distribution ofohesins along chromosomes was initially monitoredy modifying existing protocols for chromatin immu-oprecipitation (ChIP) in S. cerevisiae, in which theNA immunoprecipitated with cohesin subunits was

adiolabeled for use as a probe (Blat and Kleckner, t

999). These probes were then hybridized to a mem-rane containing an array of PCR-generated chromo-ome fragments, covering the entire length of chromo-ome III.Amajority of cohesin binding sites identifiedn this way were associated with the centromere,lthough the complex was also shown to bind specificites along the chromosome arms (Blat and Kleck-er, 1999; Tanaka et al., 1999), consistent with theiscrete foci seen in chromosome spreads (Tóth et al.,999). These binding sites were found to correlateith locally AT-rich sequences, occurring roughlyvery ,15 kb along the chromosome (Blat andleckner, 1999). This preference for AT-rich se-uences in centromeric regions was further corrobo-ated by conventional ChIP analysis, using the immu-oprecipitated DNA as a PCR template (Megee et al.,999). Although such regions are reminiscent ofARs, no correlation with the redundant motifs ofrosophila SARs was observed (Blat and Kleckner,999). However, this does not preclude the possibil-ty that SARs might indeed be binding sites forohesins, bearing in mind the differences betweenhe short, defined centromeres of S. cerevisiae andhe longer regional centromeres of other eukaryotesuch as S. pombe and Drosophila melanogasterPluta et al., 1995). Indeed, it was previously ob-erved that SMC proteins bind preferentially torosophila SARs as well as sequences containinglternating poly(dA–dT) and yeast centromere re-ions (Akhmedov et al., 1998). Interestingly, a moreven distribution of cohesin binding in hydroxyurea-rrested cells suggested that cohesins bind uni-ormly to chromosomes at the start of S phase (Blatnd Kleckner, 1999) but relocate to centromericegions later during the cell cycle so the highestevels of centromere-bound Scc1p/Mcd1p were seenn cells arrested in M phase (Megee et al., 1999).

The minimal centromere sequences required forohesin association were then deduced by artificiallynserting sequences from CEN6 into a region whichormally has low cohesin affinity (Tanaka et al.,999). Normally the insertion of additional centro-eres in this manner would create unstable dicen-

ric chromosomes, so the inserted centromeric se-uences were conditionally suppressed by placinghem under the control of a galactose-inducibleromoter. In this way it was deduced that 130 bpontaining CDEI–II–III was sufficient to confer cohe-in binding to this sequence, whereas cohesin associa-ion with CEN DNA was abolished by transcriptionrom the GAL promoter. The CDEIII sequence inarticular was shown to be sufficient for weak cohe-in association, but could be enhanced by adding 21p of CDEII (Tanaka et al., 1999). Moreover, associa-ion was abolished by various CDEIII point muta-

ions, further supporting the importance of this

ean(wtqcsciapspwc

asdaaStdtta(idsMotA1cdv

Cigpniocparc

dtwFrcoefwMdreeSosmeMptro

Ssd(dStarXtPnStowmodaIede


lement for cohesin binding (Tanaka et al., 1999), ingreement with previous findings that CDEIII wasecessary but insufficient for functional cohesionMegee and Koshland, 1999). Despite this, cohesinsere shown to differ from known centromere pro-

eins by associating strongly with adjacent se-uences as well as the centromere itself, in someases with even higher affinity for these flankingequences (Megee et al., 1999). As ectopically placedentromeres were shown to direct Scc1p/Mcd1p bind-ng to adjacent sequences which normally had lowffinity for the protein (Megee et al., 1999), it is thereforeossible that the centromere primarily contributes toister chromatid cohesion by directing the cohesin com-lex to AT-rich sequences in the immediate vicinity. Itill be interesting to see whether similar patterns of

ohesin association are found in other organisms.

THE SEPARATION OF SISTERS

How is the cohesin-mediated cohesion dissolved tollow separation of sister chromatids during mito-is? Both Scc1p/Mcd1p and Scc3p were shown toissociate from chromosomes at the metaphase tonaphase transition (Michaelis et al., 1997; Tóth etl., 1999), leaving behind the bulk of Smc1p andmc3p cohesin subunits which associate longer withhe chromosomes (Tanaka et al., 1999). Although theissociation of Scc1p/Mcd1p was known to depend onhe anaphase promoting complex (APC), this dissocia-ion could be prevented by expression of a nondegrad-ble version of Pds1p but not by other APC inhibitorsCohen-Fix et al., 1996; Michaelis et al., 1997). Thismplied that ubiquitination by the APC was notirectly responsible for Scc1p/Mcd1p destruction anduggested that Pds1p might somehow prevent Scc1p/cd1p dissociation. This was confirmed by deletion

f the pds1 gene, allowing sister chromatid separa-ion and Scc1p/Mcd1p dissociation in the absence ofPC function (Ciosk et al., 1998; Yamamoto et al.,996). However, the premature separation of sisterhromatids in scc1/mcd1 mutants contrasted with theelayed separation in pds1 mutants, inferring the in-olvement of other factors in the control of anaphase.Additional insights were provided by studies ofut2p, an S. pombe orthologue of the anaphase

nhibitor Pds1p. This protein was shown to be de-raded by the APC and copurified with Cut1p, arotein maintained throughout the cell cycle (Fu-abiki et al., 1996a,b, 1997). Similarly, Pds1p was

mmunoprecipitated with Esp1p (the S. cerevisiaerthologue of Cut1), a protein required for sisterhromatid separation (Ciosk et al., 1998). As the tworoteins in the complex had antagonistic effects onnaphase, the Esp1p and Cut1p were named ‘‘sepa-ins’’ while their inhibitors (Cut2p and Pds1p) were

alled ‘‘securins.’’ Moreover, since Pds1p was not i

egraded by Esp1p, it appeared that securins inhibithe separins by binding to them but this inhibitionas removed by APC-mediated proteolysis (Cohen-ix et al., 1996). The activity of Esp1p was thenevealed when its overexpression permitted sisterhromatid separation in the presence of Pds1p (Ci-sk et al., 1998). As sister chromatid separation insp1 mutants was also shown to be prevented by aailure of Scc1p/Mcd1p degradation, the separinsere therefore considered to be responsible for Scc1p/cd1p dissociation. This role was confirmed by

emonstrating that Scc1p/Mcd1p cleavage and itsesultant dissociation from chromatin occurred inxtracts from cells overexpressing Esp1p but not insp1 mutant extracts (Uhlmann et al., 1999). Thecc1p/Mcd1p cleavage sites were then identified andverexpression of a cleavage-resistant protein washown to prevent sister chromatid separation (Uhl-ann et al., 1999). In conclusion, the available

vidence seems to suggest that dissociation of Scc1p/cd1p from sister chromatids in S. cerevisiae de-

ends on cleavage mediated by Esp1p, which isransported to the spindle by its inhibitor Pds1p butemains inactive until the APC triggers proteolysisf Pds1p at the metaphase–anaphase transition.As the ability of S. cerevisiae cell extracts to cleave

cc1p/Mcd1p correlated with the levels of Esp1p, theimplest explanation is that Esp1p is the proteaseirectly responsible for Scc1p/Mcd1p degradationUhlmann et al., 1999). However, until it can beemonstrated that purified Esp1p is sufficient forcc1p/Mcd1p cleavage in vitro, one cannot excludehe alternative possibility that this protein mightctivate another protease instead. Furthermore, itemains to be seen if cleavage of the human andenopus Rad21p orthologues can be suppressed by

he recently characterized functional homologue ofds1p in Xenopus (Zou et al., 1999). Certainly, theeed to remove PW29 (the murine orthologue ofcc1p/Mcd1p) to allow separation of sister chroma-ids has been demonstrated by the metaphase arrestf mitotic cells when a PW29–GFP fusion proteinas overexpressed (Darwiche et al., 1999). However,urine PW29 protein levels appear constant through-

ut the cell cycle, whereas Scc1p/Mcd1p levels peakuring S phase and decline thereafter (Darwiche etl., 1999; Guacci et al., 1997; Michaelis et al., 1997).t therefore seems that anaphase occurs by a differ-nt mechanism in vertebrates, involving not justegradation of the cohesin complex but also itsxclusion from the chromosomes.

CONNECTIONS BETWEEN COHESIONAND CONDENSATION

Considering the involvement of complexes contain-

ng SMC proteins in both sister chromatid cohesion

atdtcdppttmstcdtpssi(aStctpmB1inc

hei(osdsecachrtoipspep

bt1pocXlMtscli

ipbSscLttdpopcaAettFinttrtS

idrMnir1rh


nd chromosome condensation, is there any struc-ural interrelationship between these processes? Toate, none of the non-SMC subunits have been foundo be shared between the cohesin and condensinomplexes, which would seem to indicate indepen-ent evolution of these two SMC-containing com-lexes. Clearly condensation is a prerequisite forroper segregation of sister chromatids, ensuringhat the entire chromosome is accurately packagedo avoid such hazards as sister chromatid entangle-ent and cleavage of trailing chromatin at cytokine-

is. A model depicting the deposition and activity ofhe cohesin and condensin complexes during the cellycle is given in Fig. 3. In S. cerevisiae it has beenemonstrated by FISH that chromosome condensa-ion also depends on the cohesin subunit Scc1p/Mcd1rotein (Guacci et al., 1997). Similarly, the dispersedtaining of nuclear material and stretched chromo-omes in S. pombe rad21 mutants may reflect itsnvolvement in chromosome condensation as wellBirkenbihl and Subramani, 1995; Tatebayashi etl., 1998). It has been proposed that placement of thecc1p/Mcd1 protein at the newly replicated chroma-ids provides an attachment site for recruitment ofondensation proteins, suggesting a possible explana-ion for the mirror symmetrical, helically foldedattern often observed in the condensed sister chro-atids of vertebrate cells (Baumgartner et al., 1991;oy de la Tour and Laemmli, 1988; Rattner and Lin,985). Nevertheless, the condensation defects result-ng from mutations affecting cohesin subunits areot as severe as those affecting components of theondensin complex (Strunnikov et al., 1995).By contrast, the vertebrate cohesins do not seem to

ave an effect on chromosome condensation (Losadat al., 1998) and neither hCAP-E nor hCAP-C aremmunoprecipitated with either hSMC1 or hSMC3Schmiesing et al., 1998). Furthermore, the bindingf condensins to chromatin and chromosome conden-ation itself seem unaffected by cohesin immuno-epletion in Xenopus egg extracts and cohesinsimilarly bind to chromatin in condensin-depletedxtracts (Losada et al., 1998). The differences inohesin and condensin behavior between vertebratesnd yeast may reflect the relatively small amount ofondensation occurring in yeast compared to that inigher eukaryotes (Guacci et al., 1994). Thus, theoles of the cohesin and condensin complexes appearo have become more specialized in higher eukary-tes so that these complexes associate and dissociatendependently. Furthermore, no cell-cycle-specifichosphorylation has been observed for the cohesinubunits in Xenopus, unlike the mitosis-specific phos-horylation of condensin complex members (Losadat al., 1998). On the other hand, the S. pombe Rad21

rotein is initially phosphorylated in G1/S, followed c

y hyperphosphorylation in G2 which is maintainedhroughout mitosis (Birkenbihl and Subramani,995). Strikingly, the cohesin complexes from Xeno-us egg extracts resemble condensins as they alsoccur as two versions with different sedimentationoefficients, in which the 9S form is a heterodimer ofSMC1 and XSMC3 while the 14S form contains at

east three additional subunits (including the Scc1p/cd1p orthologue) (Losada et al., 1998). This struc-

ural similarity between cohesins and condensinstrongly suggests that they may have evolved from aommon ancestor (albeit independently), particu-arly considering that a single SMC protein may benvolved in both processes in bacteria.

A further link between cohesion and condensationn budding yeast is revealed by analysis of the Trf4rotein (topoisomerase I-related function), whichinds to both Smclp and Smc2p (Castaño et al., 1996;trunnikov et al., 1993) and is required for chromo-ome segregation (Castaño et al., 1996) and rDNAhromosome condensation (Castaño et al., 1996).ikewise, an additional link between the condensa-

ion and cohesion machinery has been revealed byhe interaction of either Smc1p or Smc2p withifferent coiled-coil domains of the human HEC1rotein (highly expressed in cancer) and Tid3p, itsrthologue in S. cerevisiae (Zheng et al., 1999). Thisrotein is required to prevent haphazard sisterhromatid segregation in both organisms (Chen etl., 1997; Zheng et al., 1999) and to repress theTPase activity of the 26S proteasome subunit (Chent al., 1997), suggesting possible roles in regulatinghe destruction of Scc1p/Mcd1p or even controllinghe ATPase activity of SMC-containing complexes.urthermore, as Tid3p was previously shown to

nteract with a protein required for meiotic recombi-ation and synaptonemal complex formation (DMC1),his suggests a possible role for HEC1 in extendinghe activities of SMC proteins to recombinationepair during meiosis (Dresser et al., 1997). Clearly,he biochemical effects of HEC1 interaction withMC proteins demand further study.

COHESIN PROTEINS AND MEIOSIS

The subunits of the cohesin complex also sharemportant links with proteins required for cohesionuring meiosis, as shown by the essential meioticoles of Smc3p and Rec8p (a paralogue of Scc1p/cd1p) (Klein et al., 1999; Parisi et al., 1999; Wata-

abe and Nurse, 1999). The rec8 gene was originallydentified in a screen for S. pombe mutants witheduced meiotic recombination (Ponticelli and Smith,989) and the encoded protein was shown to beequired for sister chromatid cohesion and pairing ofomologous chromosomes during meiosis I (Kraw-

huk et al., 1999; Krawchuk and Wahls, 1999; Mol-

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ar et al., 1995). Synthesis of Rec8p was specific toeiosis (unlike other cohesins) and deletion of the

ec8 gene resulted in equational rather than reduc-ional chromosome segregation (Lin et al., 1992;atanabe and Nurse, 1999). Conversely, the replace-ent of Rad21p by Rec8p during mitosis produced a

emarkable shift in the pattern of chromosome segre-ation from equational to reductional. Nonetheless,he ability of the Rad21p protein to rescue thenviability of rec8 mutant spores showed that theseroteins share common functions. Moreover, as theohesin cleavage sites appear to be uniquely con-erved between Scc1p/Mcd1p in S. cerevisiae, Rad21pn S. pombe, and the Rec8 proteins in both organ-sms, it seems likely that their cleavage may beimilarly mediated by separins (Uhlmann et al.,999).The Rec8 protein was originally believed to be

nvolved in early steps of meiotic recombination,ased on its early appearance and disappearanceLin et al., 1992). In agreement with this, rec8utants exhibited defective linear elements (axial

ore-like structures formed in place of tripartiteynaptonemal complexes during meiotic prophase in. pombe) which were shorter and thicker than inild-type cells (Molnar et al., 1995; Parisi et al.,999). Likewise, both Rec8p and Smc3p (which areequired for meiotic chromatid cohesion in S. cerevi-iae) are essential for the formation of synaptonemalomplexes and axial elements (Klein et al., 1999).owever, neither protein is required for the forma-

ion of double-strand breaks, implying that Rec8pnd Smc3p are needed to maintain cohesion so thatuch lesions may be repaired (Klein et al., 1999).The S. pombe Rec8p protein was tightly associatedith numerous chromosomal foci during prophase ofeiosis I and was globally distributed around the

entromeric regions, whereas Rad21p was predomi-antly found near the telomeres (Parisi et al., 1999;atanabe and Nurse, 1999). Just as Rad21p is

hosphorylated in mitosis, Rec8p also underwenthosphorylation from prophase onward. Althoughhe level of a Rec8–GFP fusion detected by Westernlotting declined between the successive meioticivisions, the protein persisted beyond meiosis I andemained tightly associated with centromeric hetero-hromatin. Similarly, Rec8p and Smc3p in S. cerevi-iae colocalized in a continuous line along the longi-udinal axis of pachytene chromosome cores whilecc1p was restricted to discrete foci (Klein et al.,999). The Rec8 protein levels were highest at theime of premeiotic DNA replication but decreasedfter pachytene and disappeared after anaphase IIKlein et al., 1999). Correspondingly, both Rec8p andmc3p disappeared from the chromosome arms after

achytene but persisted near the centromeres after s

he separation of homologous chromosomes duringhe first meiotic division, until anaphase of meiosisI (Klein et al., 1999). Interestingly, the Rec8p local-zation pattern in both yeasts is similar to that ofOR1 (a component of the lateral elements of synap-

onemal complexes in rodent spermatocytes), sugges-ive of a role in synaptonemal complex formationDobson et al., 1994; Lammers et al., 1994; Yuan etl., 1998).It will be interesting to see if SMC3 and indeed

MC1 proteins are involved in maintaining meioticohesion in other organisms, as suggested by a highate of expression in rodent ovaries and testesShimizu et al., 1998; Stursberg et al., 1999). Theeiotic function of cohesin proteins seems to be

ighly conserved among eukaryotes as the DIF1determinate, infertile1) gene of Arabidopsis, encod-ng an orthologue of Rec8p, was similarly shown toe essential for meiotic chromosome segregation andence fertility (Bhatt et al., 1999). In addition, thebility of the human Rec8 protein to partially comple-ent the reduced spore viability of S. pombe rec8utants suggests at least some conservation of

unction (Parisi et al., 1999).

SMC PROTEINS AND DNA REPAIR

Anumber of the proteins involved in sister chroma-id cohesion have also been shown to play criticaloles in recombinational repair. For example, bothMC1 and SMC3 may have a role during interphases part of the bovine recombination protein complexC-1, in which they are complexed with DNA ligase

II, DNA polymerase e, and a DNA structure-specificndonuclease (Jessberger et al., 1996a,b). Similarly,ad21p of S. pombe was implicated in the repair ofouble-strand DNA breaks in irradiated cells inddition to being essential for mitotic growth (Birken-ihl and Subramani, 1992, 1995; Tatebayashi et al.,998).However, a specific role in DNA repair is demon-

trated by members of a further subgroup of theMC family, first identified in S. pombe through thenalysis of rad18 mutants. A temperature-sensitiveutant was shown to be hypersensitive to both UV

nd g-irradiation and also exhibited reduced rates ofemoval of UV photoproducts compared to wild-typeells. However, no significant difference in endonucle-se activity was observed between extracts fromad18 cells and wild-type cells, suggesting thatad18p might be involved in repair of DNA damagey facilitating genetic recombination. This was sub-equently confirmed by assaying the ability of cellso repair double-stranded DNA breaks using pulse-eld gel electrophoresis (Verkade et al., 1999). Corre-pondingly, severely reduced levels of intrachromo-

omal homologous recombination were demonstrated

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n an Arabidopsis mim mutant (an orthologue ofad18) (Mengiste et al., 1999).Immunostaining of cells revealed that the protein

s found in the chromatin compartment of the S.ombe nucleus, as expected for a protein directlyngaged in DNA repair (Verkade et al., 1999). Aad18 mutant was completely suppressed by excessopies of brc1 (encoding a BRCT domain proteinequired for proper chromosome condensation andegregation) but was synthetically lethal in combina-ion with mutations in brc1, fin1 (encoding a kinasehich induces chromatin condensation), or topoisom-rase II, suggesting that the ability of Rad18p toepair DNA lesions might be related to a role inrderly chromosome condensation. It will be interest-ng to see whether Rad18p shares the ability of somether SMC proteins to directly promote strand ex-hange (Jessberger et al., 1993, 1996a,b; Sutani andanagida, 1997).The deletion of RAD18 and RHC18 (the S. cerevi-

iae orthologue) showed that the gene is essential forroliferation in both species (Lehmann et al., 1995).he mitotic defect was further characterized by the

solation of an additional temperature-sensitivead18 mutant (Verkade et al., 1999). After irradia-ion, many cells underwent cytokinesis in the ab-ence of completed chromosome segregation, result-ng in cells with nuclei stretched along the divisionlane and bisected by septa. Such aberrant mitosesn spite of unrepaired DNA lesions also implied a roleor Rad18p in maintaining the G2 DNA damageheckpoint. This was verified by the failure of twoifferent rad18/top2 double mutants to arrest in G2,hereas inhibition of topoisomerase II activity nor-ally produces such an arrest (Hartwell and Wein-

rt, 1989). However, Rad18p is not required fornitiation of the checkpoint, as shown by normalhosphorylation of the Chk1 protein kinase (thenal element in the signaling cascade activated byhe G2 DNA damage) in rad18 cultures (Verkade etl., 1999). It is therefore possible that Rad18p activ-ty is induced posttranscriptionally by this G2 check-oint and the continued activity of this proteinaintains the arrest until the damage is repaired.hus, the Rad18 subfamily of SMC proteins appearso have multiple functions in response to DNAamage, signaling the persistence of unrepairedesions in DNA and repairing them through a role inecombination-mediated repair.

MOONLIGHTING IN THE BASEMENT MEMBRANE?

Perhaps the most surprising result concerningMC proteins to date is a possible additional roleutside the cell. This idea is based on the identifica-ion of an extracellular, secreted proteoglycan, known

s bamacan (basement membrane-associated chon- t

roitin sulfate proteoglycan), as an SMC molecule.hen rat bamacan was cloned (Wu and Couchman,

997), the authors noted that the sequence bore notructural similarity with any chondroitin/dermatanulfate proteoglycan reported at that time. However,hey noticed that bamacan and SMC proteins fromeveral diverse organisms shared a similar five-omain structure. Unfortunately, as none of theMC proteins used in their comparison belonged tohe SMC3 subclass, the overall sequence homology ofamacan to other SMC proteins was found to be loweven when compared with those of vertebrate ori-in). The human orthologue of SMC3 was cloned theollowing year and was initially named HCAP (hu-an chromosome-associated polypeptide). The au-

hors commented that this SMC protein shared 98%mino acid sequence identity with the published ratamacan protein sequence (Shimizu et al., 1998).inally, the murine orthologue of the rat bamacanas cloned and identified as a member of the SMC3rotein subfamily, as the protein showed the sameevel of homology to the bovine SMC3 as it did to theat bamacan (Ghiselli et al., 1999). Meanwhile, anndependent group had succeeded in cloning the

urine homologue of SMC3, known as mSMCDDarwiche et al., 1999). However, at the time ofriting both groups were seemingly unaware of justow close bamacan really was to SMC3. When theDNA sequences for the murine SMC3 and murineamacan are aligned, the corresponding predictedrotein sequences are 100% identical. In other words,t appears that the mouse SMC3 is the same mol-cule as a component of the extracellular basementembrane.As the murine bamacan was cloned by using the

at bamacan sequence to BLAST the dbEST dataase, the true significance of the similarity hinges onow reliably the rat bamacan was cloned. The ratamacan was originally isolated (Wu and Couch-an, 1997) by screening a rat yolk sac carcinoma

DNA expression library with a polyclonal antise-um raised against a pool of purified proteoglycansrom the murine Engelbreth–Holm–Swarm tumoratrix (Couchman et al., 1996). To confirm the

dentity of this clone, rabbit antibodies were raisedgainst two nonoverlapping fusion proteins encodedy subclones of the bamacan cDNA and both of thesentibodies were shown to recognize the same proteiny immunoblotting as the original antiserum. One ofhese antisera also stained extracellular matrix inissue sections, as did the original antibody. This istrong evidence that a protein better known for itsole in chromosome mechanics has a very unpre-icted extracellular localization. Antibodies to theurine SMC3 were generated against a peptide in

he C-terminal ATP-binding domain (Darwiche et

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l., 1999); these antibodies demonstrated that theurine SMC3 binds to interphase chromatin and

issociates from it at the onset of mitosis, in agree-ent with previous studies with the Xenopus ortho-

ogues (Losada et al., 1998). It would therefore berucial to see whether these antibodies used onouse tissue sections independently reproduce the

attern of staining observed with the antibodies toat bamacan.Naturally, one might wonder what a protein in-

olved in chromosome dynamics might be doing inhe basement membrane, outside the cell. Althought is not unheard of for the same protein molecule toave more than one distinct action (Jeffery, 1999),one of the previously recognized functions of SMC3how any obvious connection with a role outside theell. However, one clue to how SMC proteins could benvolved in stabilizing the extracellular matrix ofasement membranes may be provided by compari-on with the laminins (Timpl and Brown, 1996).hese molecules also have coiled-coil domains, inhis case mediating heterotrimerization between the, b, and g chains. In addition, the globular laminin-terminal domains mediate Ca21-dependent poly-erization to yield quasihexagonal networks. These

aminin networks are finally anchored in the base-ent membrane by integrin and dystroglycan recep-

ors. One may conjecture that SMCs might also formore complex networks if they are secreted in suffi-

iently high concentration, forming chains as a re-ult of interactions between the terminal ATPaseomains of adjacent molecules. Alternatively, it isossible that secreted SMC3/bamacan may fit into aatrix through chondroitin sulfate side chains, inuch the same way that perlecan (a heparan sulfate/

ermatan sulfate proteoglycan) interacts with theeparan sulfate binding site in the C-terminal LGodules of laminin a chains. An additional question

oncerns how an SMC protein might actually reachhe basement membrane. Sequence analysis of theMC proteins using available data for nuclear local-

zation signals (NLS) and NES reveals that eachMC may contain potential NLS and NES se-uences, of which at least one candidate NES haseen shown to be functional (Sutani et al., 1999).Indeed, SMC3 is not the only nuclear protein that

eems to be playing an additional role outside theell. For example, histone H1 has been shown to acts a binding protein for thyroglobulin at the cellurface of macrophages, mediating thyroglobulinndocytosis (Brix et al., 1998), while titin, a constitu-nt of muscle sarcomeres, has also been proposed toe a component of Drosophila chromosomes (Machadot al., 1998). Another recently identified basementembrane-associated proteoglycan with candidate

ES and NLS motifs, known as leprecan (Wassen- h

ove-McCarthy and McCarthy, 1999), may possiblylay a role in chromosomal dynamics as it sharesore than 36% identity and 43% similarity with the

ynaptonemal complex protein SC56 along a stretchf 343 amino acids. In conclusion, the surprisingiscovery that an SMC protein may have a roleutside the cell, quite apart from a fundamental rolen various aspects of chromosomal dynamics, adds togrowing list of moonlighting proteins.

FUTURE PROSPECTS

The understanding of chromosome structure andehavior has been greatly enriched by the findingsade over the past few years. It is already clear that

he SMCs are important for chromosome cohesion,hromosome condensation, dosage compensation, andecombination repair. The original eukaryotic sub-amilies of SMC1, SMC2, SMC3, and SMC4 haveeen joined by a fifth branch, the Rad18 subfamily.ven more exciting is the discovery and analysis of

he single SMC within prokaryotes, leading to thendeniable conclusion that the SMCs are conservedot only in structure but also in function. The evo-

ution of the single SMC to a family constituting fiveubfamilies is certainly a matter of intrigue, as is theossible extracellular existence of an SMC protein.The Xenopus in vitro extract system, coupled with

owerful genetics in S. cerevisiae and S. pombe, hasredominantly contributed to the identification ofhese molecules and their associated proteins. Studyf the SMC proteins and the complexes and pro-esses in which they take part has not only illumi-ated the significant degree to which certain compo-ents and mechanisms are conserved, but alsoighlighted provocative questions for future study.ery likely, differences between single-celled organ-

sms and multicellular creatures will be elucidatednd with time clarified. What is clearly missing fromhe studies published to date is an analysis of theseomponents in a multicellular organism amenable toevelopmental, genetic, and cytological approaches.he identification of the genes for SMCs and associ-ted proteins has been greatly facilitated by therosophila genome project. This is currently leading

o the identification of mutations in these genes andxploitation of the ability to examine these proteinst different times of development, in different tis-ues, in different types of cell cycles (e.g., rapid,ynchronized early embryonic cycles lacking G1 and2 phases versus more normal cell cycles), and inifferent types of chromosomes (e.g., diploid versusiant, banded polytene chromosomes). There is nooubt that the future years will be as rich foresearch and progress into understanding the funda-ental questions of chromosome structure and be-

avior as the past years have been.

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We thank members of the Heck and Earnshaw labs for stimulat-ng discussions concerning chromosome organization and dynam-cs. We also acknowledge Soren Steffensen, Paola Coelho, andlaudio Sunkel at the University of Porto with whom we have anngoing, fruitful collaboration into the analysis of SMCs andartners in fruit flies. M.M.S.H. is a Senior Research Fellow in theasic Biomedical Sciences, funded by the Wellcome Trust. N.C. isPh.D. student supported by a Darwin Trust Prize Studentship.

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