Cohesin: Its Roles and Mechanisms - Haering Lab · 2014-08-12 · ANRV394-GE43-22 ARI 10 October 2009 10:50 INTRODUCTION The segregation of sister chromatids to oppo-site poles of

ANRV394-GE43-22 ARI 10 October 2009 10:50

Cohesin: Its Rolesand MechanismsKim Nasmyth1 and Christian H. Haering2

1Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom;email: [email protected] Molecular Biology Laboratory, 69115 Heidelberg, Germany;email: [email protected]

Annu. Rev. Genet. 2009. 43:525–58

First published online as a Review in Advance onAugust 24, 2009

The Annual Review of Genetics is online atgenet.annualreviews.org

This article’s doi:10.1146/annurev-genet-102108-134233

Copyright c© 2009 by Annual Reviews.All rights reserved

0066-4197/09/1201-0525$20.00

Key Words

chromosome segregation, sister chromatid cohesion, mitosis, meiosis,structural maintenance of chromosomes (Smc), ABC ATPase

AbstractThe cohesin complex is a major constituent of interphase and mitoticchromosomes. Apart from its role in mediating sister chromatid co-hesion, it is also important for DNA double-strand-break repair andtranscriptional control. The functions of cohesin are regulated by phos-phorylation, acetylation, ATP hydrolysis, and site-specific proteolysis.Recent evidence suggests that cohesin acts as a novel topological devicethat traps chromosomal DNA within a large tripartite ring formed byits core subunits.

525


INTRODUCTION

The segregation of sister chromatids to oppo-site poles of the cell during mitosis is not onlyone of the most dramatic events in the life ofcells but also one of the most important. SisterDNAs produced during S phase must be seg-regated to opposite sides of the cell before itdivides. This is a remarkable feat, consideringthat the uncoiled DNA of a single human cellwould stretch for 2 m and, as a random coil ofnaked DNA, would occupy a volume of 2.6 ×107 μm3, which is several orders of magnitudelarger than the cells themselves. How do mitoticcells organize their DNA into sausage-shapedchromosomes only a few micrometers long?How do they avoid tangling sister DNAs to-gether, instead organizing them in largely sep-arate domains (chromatids) lying side by side?And how do identical chromatids know thatone sister has to move to one cell pole whilethe other sister has to move to the oppositepole, such that cytokinesis can eventually gen-erate two genetically equal daughter cells? Keyto these processes is the physical connection ofsister DNAs, tenuous along chromosome armsbut more intimate at centromeres. This sisterchromatid cohesion is not merely pretty to lookat, it is essential for setting up the connectionsbetween sister kinetochores and mitotic spindlemicrotubules so that sisters are pulled in oppo-site directions. Loss of sister chromatid cohe-sion then triggers segregation of chromatids atthe metaphase-to-anaphase transition. A mul-tisubunit protein complex named cohesin isresponsible for cohesion between sister chro-matids. This review focuses on the molecularmechanisms of cohesin function.

WHY IS SISTER CHROMATIDCOHESION IMPORTANT?

One of the great mysteries of chromosomesegregation is how cells ensure that sister kine-tochores attach to microtubules that extend toopposite spindle poles, a process known as bior-ientation. It has long been supposed, but neverproven, that kinetochores form rather rigid

polar structures, that sister chromatid cohesionensures that sister kinetochores are held back toback with their microtubule-attaching surfacesfacing in opposite directions, and that attach-ment of one surface to stiff microtubules fromone pole would per se preclude attachment ofthe sister to the same pole. As first recognizedby Nicklas (123, 124), back-to-back geometrycannot explain how cells ensure that the mater-nal and paternal kinetochores of bivalent chro-mosomes attach to microtubules extending toopposite poles during meiosis I. He suggestedthat biorientation during meiosis I is achievedinstead by a process of error correction. Morespecifically, kinetochore microtubule connec-tions are unstable unless they generate tension,which only occurs when maternal and paternalkinetochores are pulled in opposite directions.Meiotic sister chromatid cohesion is essentialnot so much to hold kinetochores in the cor-rect orientation but rather to resist the tendencyof microtubules to tear the bivalent apart andthereby to create the tension necessary to sta-bilize correct attachments. The same principlemay also apply to mitotic cells.

Once biorientation has been achieved, de-struction of sister chromatid cohesion, anequally important process, must occur to ini-tiate the separation of sister chromatids atthe metaphase-to-anaphase transition. Cohe-sion destruction is inhibited by “lagging” chro-mosomes, specifically those that have not yetbioriented, in a process known as the spindleassembly checkpoint (SAC) (121).

WHAT HOLDS SISTERCHROMATIDS TOGETHER?

The first molecular explanation for sister chro-matid cohesion was inspired by the realizationthat DNA replication produces sister DNAsthat are wound around one another. It was pro-posed that decatenation mediated by Topoiso-merase II (Topo II) is a regulated process thatis not completed until all chromosomes biori-ent (115). An opportunity to test this idea arosewith the remarkable discovery that additionof a 150-bp centromere sequence to circular

526 Nasmyth · Haering


plasmid DNAs containing a replication originimproves markedly the fidelity of their segre-gation in yeast cells (16). Gel electrophore-sis demonstrated that minichromosomes withincells that had replicated their DNA and arrestedin a mitotic state due to a spindle poison suchas nocodazole (which triggers the SAC) werealmost exclusively monomeric (84). In otherwords, Topo II had already completed its task,and decatenation had been fully achieved. Theconclusion was that something other than DNAintertwining holds these particular sister DNAstogether and that this unknown mechanismmight be mediated by “one or more interest-ing proteins” (84, p. 1715).

Proteins Required for SisterChromatid Cohesion

The search for such proteins intensified withthe finding that destruction of sister chromatidcohesion at the metaphase-to-anaphase transi-tion is triggered by a ubiquitin protein ligasenamed the anaphase-promoting complex or cy-closome (APC/C) (68, 76, 164). Genetic studiesrevealed the first likely candidates (46, 108). Weknow from these and subsequent studies thatsister chromatid cohesion requires the follow-ing components.

1. A core cohesin complex containing twoSmc proteins, Smc1 and Smc3, and twonon-Smc proteins, Scc1 (also known asMcd1 or Rad21) and Scc3 (known inmammalian cells as SA1 and SA2). Allcomponents of the cohesin complex areessential for maintaining sister chromatidcohesion in postreplicative yeast cells.

2. An accessory protein composed of HEATrepeats named Pds5 that binds moreloosely to the cohesin complex but isfound at similar locations on the genomeas cohesin.

3. A separate complex containing the HEATrepeat Scc2 protein (known as Nipbl inmammalian cells) and the TPR repeatScc4 protein. This complex is essential forcohesin’s association with chromosomes.It is necessary to establish cohesion but

not to maintain it after DNA replicationhas been completed.

4. An acetyl transferase named Eco1 (Esco1and -2 in mammalian cells), which, likethe Scc2/4 complex, is needed for the es-tablishment of cohesion during S phasebut not for its subsequent maintenance.The vertebrate Sororin protein may alsofall into this category.

In addition to these proteins (Table 1),which are essential for mitosis, many others arenecessary merely to improve the efficiency ofsister chromatid cohesion. Budding yeast mu-tants lacking these proteins are viable despitehaving high rates of chromosome missegre-gation. Their patterns of “synthetic lethality”have formed the basis for dividing many of thenonessential cohesion proteins of Saccharomycescerevisiae into two groups (207). The first groupincludes the putative DNA helicase Chl1, afactor known as Ctf4 that forms a complexwith various DNA-replication proteins (e.g.,DNA polymerase α, Mcm and GINS com-plexes, Cdc45, Mrc1, Tof1, and Csm3) (33), anda complex formed between the Tof1 and Csm3proteins that associates with replication forksand regulates their response to being stalled(132, 207). The second group includes Mrc1(also associated with replication forks) (73) anda Replication Factor C (RFC) complex, whoseCtf1 subunit has been replaced by the Ctf1-likeprotein Ctf18 and contains the additional sub-units Dcc1 and Ctf8 (104). Most of these non-cohesin proteins are thought to contribute tosister chromatid cohesion during DNA replica-tion. It was recently suggested that both ORC(152) and condensin (90) have a role in main-taining cohesion in postreplicative cells. In thecase of ORC, the possibility that the loss of co-hesion stems from defects during DNA repli-cation has not been ruled out.

Dissolution of SisterChromatid Cohesion

The destruction of sister chromatid cohesiontakes place in two phases. The first commencesduring prophase. Cohesin at centromeres is

www.annualreviews.org • Roles and Mechanisms of Cohesin 527


Table 1 Genes encoding cohesin subunits and regulatory proteins

Cohesin complexes

Accesssory proteins

SMCsubunit

SMCsubunit

kleisinWHD subunit

HEAT repeatsubunit

HEAT repeatassociated

Saccharomyces cerevisiae

Schizosaccharomyces pombe

SMC1 SMC3

Caenorhabditis elegans

Drosophila melanogaster

Encephalitozoon cuniculi

Xenopus laevis

MCD1 = SCC1IRR1 = SCC3 PDS5

psm1 + psm3+rad21+ psc3 +

pds5+

him-1 smc-3coh-1scc-1

mit smc1a rad21

scc-3 evl-14

SMC1 CapRad21

ECU04_0930

pds5

ECU04_1370

Homo sapiensSMC1A

SMC3RAD21

REC8

smc3smc1bmei

stag1 stag2 pds5a pds5b

mit

mei

mit

mei

PDS5A PDS5BSMC1B

STAG1 STAG2

STAG3

SA SA-2

mit

mei

rec11+rec8 +

coh-3rec-8

c(2)M

REC8

mit

mei

mit

mei

ECU09_1910 ECU04_0870

Loading complex Cohesion establishment/release Separase Securin

Saccharomyces cerevisiae

Schizosaccharomyces pombe

SCC2 SCC4

Caenorhabditis elegans

Drosophila melanogaster

Encephalitozoon cuniculi

Xenopus laevis

ECO1 ESP1 PDS1

mis4 + ssl3 + eso1 + cut1 + cut2 +

Nipped-B

mau-2

CG4203

NIPBL

ECU07_0350

Homo sapiens

kiaa0892

KIAA0892

nipbl esco1

pqn-85

ESCO1 ESCO2

sep-1 ify-1

RAD61

WAPAL ESPL1 PTTG1

wapal espl1

wpl1 +

san eco pimSse thrwapl

wapl-1F08F8.4

???

(LOC398156)

rec8

Gene names of mitosis (mit) or meiosis (mei) specific Smc, α-kleisin, or HEAT repeat subunits of cohesin complexes andproteins that regulate cohesin’s association with chromosomes or establishment or release of cohesion (59, 120, 127, 131)according to Saccharoymces Genome Database, S. pombe GeneDB, WormBase, Flybase, Xenbase, or HGNC.

spared this fate, which presumably contributesto a characteristic feature of mitotic chromo-somes, namely their central (centromeric) con-striction. The second phase involves separase, ahighly conserved CD clan thiol protease, whosecleavage of Scc1 removes cohesin from chro-mosomes and triggers (at least in yeast) sisterchromatid disjunction (130, 185, 187, 194).

HOW COHESIN CONNECTSSISTER CHROMATIDS

The Structure of Cohesin

At the heart of the cohesin complex is a het-erodimer formed between its Smc1 and Smc3subunits. Each subunit, composed of a 50-nm-long intramolecular antiparallel coiled coil,

forms a rod-shaped protein with a globu-lar “hinge” domain at one end and an ATPnucleotide-binding domain (NBD) of the ABCfamily at the other (50, 57, 109). Because ofthe antiparallel nature of their coiled coils, eachSmc NBD is built from two halves, one fromC-terminal and the other from N-terminalamino acids, whereas the hinge domain is en-coded by amino acids from the center of thepolypeptide. Heterotypic interactions betweenthe hinge domains of Smc1 and Smc3 lead to theformation of V-shaped Smc1/3 heterodimerswith an Smc1 NBD at the end of one coiled-coil arm and an Smc3 NBD at the end of theother (Figure 1a). The interaction betweenSmc1 and Smc3 hinge domains is tight, witha low nanomolar KD (50). The crystal struc-ture of a homodimeric bacterial hinge from



c

Smc1Smc3

Scc1Scc3

Separase

Smc1Smc3

Scc1

ATPhydrolysis ATP binding

Scc3

CN

CN

K112/K113K112/K113K112/K113

NBDs

a b

Figure 1Structure of the cohesin complex. (a) The α-kleisin subunit Scc1 connects the nucleotide-binding domains(NBDs) situated at the apices of the V-shaped Smc1/3 heterodimer and recruits Scc3 to the complex. ATPbinding leads to NBD association, whereas ATP hydrolysis presumably drives NBD disengagement.(b) Schematic of a crystal structure of the Smc hinge dimerization domain from Thermotoga maritima(pdb 1GXL). The Smc protamers dimerize via two symmetric interfaces that are separated by a centralchannel. (c) Schematic based on the crystal structure of the Smc1 NBD bound to the C-terminal WHD ofScc1(pdb 1W1W). The second Smc1 NBD in the symmetric crystal structure was replaced by a homologymodel of the Smc3 NBD. Two molecules of ATPγS are sandwiched between the NBD interfaces. Lysineresidues 112 and 113 in the Smc3 NBD are acetylated by the Eco1 acetyltransferase. Representationsgenerated with Pymol (DeLano Scientific).

Thermotoga maritima shows that two shallowU-shaped hinge monomers interact to form atwofold symmetric torus or ring with a shapenot unlike that of DNA polymerase clamps, al-beit with a much smaller hole in the middle(Figure 1b). The most remarkable feature ofthe interaction is that the two monomers are

held together at equivalent but potentially in-dependent surfaces. Each of the two contactscan exist in the absence of the other becausethe structure of a second crystal form reveals adimer in which one interaction surface is dis-turbed by crystal contacts and in which thedimeric torroid is slightly twisted open. This



bipartite nature of Smc hinge-hinge contactsis curious but nevertheless highly conserved,and it applies equally to eukaryotic Smc het-erodimers, including cohesins Smc1/3. The 50-nm-long antiparallel coiled coils that emergefrom cohesin’s hinge dimerization domains areinterrupted by a couple of short conserved do-mains that may contribute to the bent shapeof cohesin’s coiled-coil arms observed in rotaryshadowed electron micrographs of vertebratecohesin complexes (1).

Like other ABC-like NBDs, the Smc1/3NBDs have a bilobed design wherein severalhelices form a rigid helical domain (HD) thatis flexibly attached to a set of β sheets con-taining the nucleotide-binding Walker A andB residues. The coiled coils of Smc proteins areconnected to the HD. The crystal structure ofthe yeast Smc1 NBD complexed with a frag-ment of Scc1 (51) closely resembles the struc-tures of Rad50 from Pyrococcus furiosus (61) andan Smc protein from T. maritima (101). LikeRad50 (61) and an ABC ATPase transporter(156), the Smc1 NBD crystallizes as a dimerin the presence of the slowly hydrolysable ATPanalog ATPγS (Figure 1c). The ABC signaturemotif at the tip of one of the HD’s helices withinone Smc1 NBD interacts with the phosphatesof a nucleotide bound to a Walker A motif in itspartner. ATPγS is thereby sandwiched betweentwo NBD domains.

Within cohesin itself, ATP is presumablysandwiched between Smc1 and Smc3 NBDs.Indeed, Smc1/3 heterodimers cooperate in hy-drolysing ATP when the Smc1 NBD is boundby Scc1 (3). Hydrolysis (but not binding) ofATP at either site is abolished by mutation ofhighly conserved Walker B glutamate residues.Cohesin complexes containing Smc1 or Smc3proteins with these mutations assemble nor-mally but cannot associate correctly with chro-matin or build sister chromatid cohesion (2).The precise function of ATP binding and/orhydrolysis remains mysterious. One possibil-ity is that ATP binding forces Smc1 and Smc3NBDs together, whereas hydrolysis permitstheir dissociation (Figure 1a), but it is cur-rently unclear how association/dissociation per

se of NBDs facilitates cohesin loading ontochromatin.

The HD and coiled coils of Smc proteinsare directly connected to a short loop (theQ loop), at one end of which is a highlyconserved glutamine residue that coordinatesthe active-site magnesium ion in a mannersimilar to that of other P-loop ATPases. Thisglutamine is thought to couple the rotation ofHD domains to changes in ATP binding andhydrolysis. Because of their proximity to thecoiled coils, changes in the rotation of SmcHD domains may in turn alter the rotation orangle of cohesin’s coiled coils as they emergefrom each NBD. The coiled-coil sequences(especially those of Smc3) are remarkablyconserved for some distance as they emergefrom the NBDs, suggesting that they have veryspecific properties. Indeed, the integrity of thissection of the coiled coil within Smc1 appearsessential because an insertion of five aminoacids inactivates cohesin (112).

A remarkable feature of cohesin’s structureis that the NBDs of Smc3 and Smc1 are boundtightly by Scc1’s N- and C-terminal domains,respectively, creating a huge tripartite ring (50).Both domains are found in members of the so-called kleisin family of proteins (147). This fam-ily includes (a) α-kleisins, orthologs of Scc1 andits meiotic variant Rec8, (b) β-kleisins, equiva-lent subunits of condensin II, (c) γ-kleisins, sub-units of condensin I, (d ) δ-kleisins, subunits ofSmc5/6 complexes, (e) ScpA-like proteins thatbind to bacterial Smc NBDs, and ( f ) MukF,which binds to the bacterial Smc-like MukBprotein. The stable cross-linking of Smc1 andSmc3 NBDs by α-kleisins is reminiscent ofthe bacterial MalK ABC-like transporter whoseNBDs are also connected through a so-calleddomain swap of their C-terminal domains.

A crystal structure of Smc1’s NBD bound toScc1’s C-terminal domain shows that the lat-ter forms a winged helix domain (WHD) thatbinds through extensive hydrophobic interac-tions to the two most C-terminal β strands ofthe Smc1 NBD (51). This interaction alters thestructure of Smc1’s NBD in a manner that isessential for ATP binding and hydrolysis (3).



The three helices, followed by two β strands ofScc1’s winged helix, correspond to the bound-aries and secondary structure predictions forthe C-terminal domains of all kleisins (147). Allpresumably bind to their Smc partners in a sim-ilar manner, and a recent analysis of the crystalstructure of MukF bound to MukB’s NBD con-firms this hypothesis (205).

Interestingly, mutation of amino acids eitherwithin the WHD or within the β strands ofSmc1 to which it binds abolishes all associationbetween Scc1 and Smc1/3 heterodimers in vivo(51). This finding implies that the WHD-Smc1interaction is responsible for recruiting Scc1 toSmc1/3, and only after this has taken place canthe N-terminal domain of Scc1 bind to Smc3’sNBD.

The interaction between Scc1’s conservedN-terminal domain and Smc3’s NBD is less wellunderstood. It was first revealed by the findingthat a polypeptide encoding the N-terminal 115amino acids of Scc1 copurifies with intact Smc3protein (but not Smc1) when coexpressed in in-sect cells (44, 50), as does the N-terminal half ofScc1’s meiotic counterpart Rec8 (44). Whetherthis interaction occurs in vivo was tested byanalyzing the fate of cohesin after cleavage ofScc1 by separase. The N-terminal Scc1 frag-ment produced by separase cleavage remains as-sociated with the C-terminal fragment becauseboth are attached to Smc1 and Smc3 NBDs,which are themselves connected by their associ-ated hinge domains. Meanwhile, simultaneouscleavage of Scc1 and Smc3’s coiled coil releasesScc1’s N-terminal cleavage fragment associatedwith Smc3’s NBD (44). Additional evidence forthe notion that Scc1’s N-terminal domain bindsto the Smc3 NBD is the finding that there isgreater fluorescence resonance energy transfer(FRET) in vivo between cyan fluorescent pro-tein (CFP) fused to the N terminus of Scc1 andyellow fluorescent protein (YFP) fused to the Cterminus of Smc3 than to YFP fused to the Cterminus of Smc1 (106).

The functional importance of the interac-tion has been tested by mutating residues withinScc1’s conserved N-terminal domain, which ispredicted to contain three helical sections and

to fold into a helix-turn-helix domain. Muta-tions in several residues within the third he-lical section abolish copurification of N- andC-terminal Scc1 fragments after cleavage ofScc1 with tobacco etch virus (TEV) protease,implying that these mutations affect the bindingof Scc1’s N-terminal domain to Smc3 (3). Thesame mutations abolish cohesin function, but inat least one case (D92K) lethality is rescued byfusing Scc1’s N terminus to the C terminus ofSmc3 but not by fusing its C terminus to the Nterminus of Smc1 (43). The dependency of theinteraction on prior binding of the Scc1’s C ter-minus to the Smc1 NBD ensures that separateScc1 molecules cannot bind to the two NBDs ofa single Smc1/3 heterodimer, which would pre-clude ring formation. The recent finding thatEco1 regulates the establishment of cohesionby acetylating of Smc3’s NBD (see section Es-tablishment of Cohesion) emphasizes the im-portance of understanding how Scc1 binds toSmc3 NBDs. It will also be important to as-certain whether or not the association is in anyway altered when Smc1 and Smc3 NBDs en-gage each other in the presence of ATP, as hasbeen suggested for the MukBEF complex (205).The picture emerging from these biochemicalexperiments, namely a gigantic tripartite ringcomplex containing Smc1, Smc3, and Scc1, isconsistent with electron micrographs of cohesincomplexes (1).

Little is known about Scc3, whereas Pds5 ispredicted to be composed almost exclusively ofHEAT repeats (122, 128). The latter’s associ-ation with the tripartite ring is clearly weakerand more salt sensitive than that of Scc3 (106,166). Nevertheless, association of both pro-teins with the Smc1/3 subunits depends onScc1 (50, 106). Scc3 binds directly to aminoacid residues within Scc1’s C-terminal separasecleavage fragment that are N-terminal to theconserved C-terminal WHD. Scc3 also bindsto Rad61 (yeast Wapl), at least in vitro (143).Whether Pds5 binds directly to Scc1 or only viaan intermediary like Rad61 is not known (143).Pds5 forms a more stable subcomplex withRad61/Wapl (88, 143). Because Rad61 is notessential for sister chromatid cohesion, whereas



Pds5 is, the latter must presumably bind cohesinindependent of Rad61/Wapl. Strangely, in vivoFRET failed to detect proximity of Pds5 to Scc1(or Scc3) but did detect a weak signal with theSmc1/3 hinge. However, it has not yet beenpossible to measure a physical interaction be-tween Pds5 and isolated hinge complexes (106).

The Ring Model

The discovery that Smc1, Smc3, and theα-kleisin Scc1 form a ring has led to the pro-posal that cohesin associates with chromosomesby trapping DNA/chromatin fibers. If so, co-hesin could hold sister DNAs together by trap-ping them both inside the same ring (50). Ring(or embrace) models of this nature come intwo flavors. The strong version holds that sisterDNAs are trapped inside a single monomericcohesin ring (Figure 2a), whereas weak ver-sions (sometimes referred to as handcuff mod-els) postulate that sister DNAs are held togetherby interactions between two different rings, onethat has trapped one DNA and a second that hastrapped its sister (Figure 2b,c).

Both weak and strong ring models holdthat cohesin grasps chromatin using a topolog-ical principle rather than physically binding toDNA or nucleosomes. Accordingly, both typesof model predict that breaking the ring at any

point should trigger cohesin’s dissociation fromchromatin and loss of sister chromatid cohe-sion. This hypothesis has been tested throughuse of TEV protease to cleave open cohesinrings. Cleavage of α-kleisin at TEV sites ei-ther at a mutated separase site (187) or else-where within its central domain (44, 130) doesindeed have this effect. Importantly, if the TEVsites are flanked by sequences encoding MP1on one side and p14 on the other—two pro-tein domains that associate with each other witha low nanomolar KD and a low off rate—thenα-kleisin cleavage no longer affects sister chro-matid cohesion (43). This finding implies thatit is not the generation of novel N or C ter-mini that compromises cohesin’s ability to holdsisters together but rather the disconnection ofN- and C-terminal domains of its α-kleisin sub-unit. Severing the coiled coil of Smc3 has beenachieved through insertion of TEV cleavagesites within regions of low coiled-coil proba-bility on both strands at positions that coincidewithin its coiled-coil arm (44). Cleavage of onlyone strand has little or no effect on cohesin’sactivity, but simultaneous cleavage triggers co-hesin’s dissociation from chromosomes (44, 69).Yet another way of opening cohesin rings thathave previously associated with chromatin is touse mutations that weaken one of their three in-tersubunit interactions. The S525N mutation

a b c

Strong ring model Weak ring models

Figure 2Sister chromatid cohesion by cohesin rings. (a) The strong version of the ring model envisages that sisterchromatids (displayed as 10-nm fibers, e.g., DNA wrapped around nucleosomes) are entrapped within asingle cohesin ring. (b) One version of a weak ring model (the handcuff model) envisages association of (bybinding to a single Scc3 subunit) two tripartite Smc1/3/Scc1 rings. (c) Another version of the weak ring modelproposes the topological interconnection of two cohesin rings, each with a single sister chromatid entrapped.



within Scc1’s C-terminal WHD causes its in-teraction with Smc1’s NBD to be sensitive tohigh temperature. If cells with this mutationare allowed to undergo DNA replication at thelow temperature and are then arrested in Mphase, subsequent incubation at the restrictivetemperature destroys sister chromatid cohesion(14). In addition, mutations disrupting eitherhinge interface weaken Smc1/3 dimerizationand greatly reduce the stability of cohesin’s as-sociation with pericentric chromatin in yeast,with lethal consequences for sister chromatidcohesion (A. Mishra & K. Nasmyth, personalcommunication).

Both weak and strong ring models predictthat once DNAs are trapped inside cohesinrings it would be impossible to substitutering components after cohesion has beenestablished without DNA escaping and sisterchromatid cohesion being lost. Investigatorsintially addressed this prediction by expressingin postreplicative cells an α-kleisin subunit thatcannot be cleaved by separase. If the noncleav-able subunit could exchange with the wild-typeprotein, then it should hinder destruction ofsister chromatid cohesion when cells activateseparase at the onset of anaphase. This does notoccur. Noncleavable α-kleisin only blocks sisterchromatid disjunction when it is expressed priorto DNA replication (51). Likewise, expressionof wild-type ring components postreplicativelyin cells with temperature-sensitive alleles can-not rescue their loss of cohesion upon transferto the restrictive temperature (93, 161, 163,190), at least in the absence of DNA damage.This is true for Scc1 and Smc1, which are corering components, but not for Scc3, which isnot part of the tripartite ring (M. Brunet & K.Nasmyth, unpublished observations).

If association between cohesin and chro-matin is primarily topological, then cohesinrings might be free to slide along chromatinfibers when these are not stably packaged intohigher order structures. Cohesin rings have adiameter between 30 to 35 nm, which is consid-erably larger than an extended 10-nm nucleoso-mal chromatin fiber. This finding led to the im-portant prediction that the association between

cohesin and a small circular minichromosomeshould be broken by cleavage not only of thecohesin ring but also of the DNA. Cohesin as-sociated with purified minichromosomes can bedetected by immunoprecipitation of minichro-mosome DNA through use of antibodies spe-cific for epitope tags attached to cohesin. Aspredicted by both types of ring model, copre-cipitation of minichromosome DNA with co-hesin is abolished either by cleaving the cohesinring with TEV protease or by linearizing theDNA with a restriction enzyme (69).

A key step has been the development ofmethods to detect sister minichromosome co-hesion in vitro (70). Extracts are first frac-tionated by differential sedimentation veloc-ity on sucrose gradients and are subsequentlyelectrophoresed under native conditions inagarose gels. G1 cells contain a single species ofminichromosome DNA, namely one that sedi-ments slowly in gradients but migrates rapidlyin agarose gels. G2- or M-phase cells, how-ever, contain an additional higher-molecular-weight (dimeric) form that sediments morerapidly and electrophoreses more slowly. Cru-cially, dimeric minichromosomes are convertedto the monomeric form by cleavage of Scc1by TEV protease. As predicted by both strongand weak ring models, linearization of co-hesed dimeric minichromosomes converts theirelectrophoretic mobility to that of linearizedmonomers.

The notion that cohesin holds sisterminichromosomes together via a purely topo-logical mechanism makes a strong prediction,namely that if the three subunit interactionsthat create the cohesin ring were cross-linked,then sister DNAs would be trapped insidea covalently circularized cohesin ring. Un-der these circumstances, they would migrateas dimers during gel electrophoresis even af-ter protein denaturation with sodium dodecylsulfate (SDS). Researchers have achieved chem-ical circularization by fusing Smc3’s C termi-nus to Scc1’s N terminus and by introduc-ing at the other two ring interfaces pairs ofcysteine residues that can be cross-linked bythe thiol-reactive cross-linkers dibromobimane



(bBBr) and bis-maleimidoethane (BMOE) (49).Either bBBr or BMOE induces cross-linkingat both interfaces in ∼30% of cohesin rings,which causes ∼30% of the DNAs from “native”dimers to form SDS-resistant dimers that mi-grate slightly more slowly than do DNA-DNAconcatemers. These dimers are actually com-posed of monomeric DNAs that are held to-gether by cohesin in a manner dependent onall four cysteine insertions and cross-linkingreagents but that are sensitive to proteolyticcleavage by TEV protease at a unique sitewithin the polypeptide interconnecting Smc3and Scc1.

Given the specificity of the cross-linking bybBBr and BMOE, there is no reason to sup-pose that putative interactions between differ-ent cohesin rings will have been cross-linked inthese experiments, and they therefore excludethe possibility that the connection between sis-ter DNAs is mediated by nontopological in-teractions between cohesin complexes associ-ated with each sister (111). The efficiency ofSDS-resistant dimer formation and its resis-tance to site specific proteolysis when createdfrom diploids expressing cleavable and non-cleavable cohesin rings are consistent with thenotion that sister DNAs are entrapped by a sin-gle monomeric ring (49). Double-ring modelsinvolving either a pair of concatenated rings,each holding a separate sister DNA (Figure 2c),or double-sized rings (formed by interconnect-ing two subunits each of Smc1, Smc3, and Scc1)are hard to reconcile with the finding that thefraction of DNAs dimerized is almost identicalto the fraction of cohesin rings circularized andnot to the square of this fraction. Neverthe-less, ultimate proof that a single cohesin ringholds sister DNAs together may require directquantification of the number of cohesin ringsassociated with dimeric minichromosomes.

If trapping of DNAs inside cohesin ringsforms the basis for sister chromatid cohesion ofreal chromosomes as well as circular minichro-mosomes, then it is clearly essential to ex-plain how this process occurs. There are threepossibilities: (a) Either the ring is assembledde novo around DNAs, or (b) DNA itself is

transiently broken and one arm of a preassem-bled ring passed through the break before itis resealed, or (c) the ring itself must be tran-siently opened. Assembly of the ring aroundDNA does not seem likely for a number ofreasons. First, cohesin complexes not associ-ated with chromatin also exist as a ring (44).These include soluble wild-type complexes aswell as complexes with mutations in Smc1 orSmc3 Walker B motifs that compromise ATPhydrolysis and cannot therefore associate stablywith chromosomes (2). Second, most cohesin inplant, animal, and fungal cells other than yeastdissociates from chromosomes during prophaseand exists as a soluble complex during mito-sis until it reassociates with chromosomes dur-ing telophase (52, 98, 167). Third, the cohesinsubunits stored in eggs that are used to estab-lish sister chromatid cohesion during cleavagedivisions are clearly present as preassembled co-hesin complexes (98, 100) that have the appear-ance of monomeric rings under the electron mi-croscope (1). The DNA-breakage model seemsinherently implausible. This leaves us with theidea that one of the three interfaces between thetripartite ring subunits is opened in a regulatedfashion and constitutes an entry gate.

Cohesin’s entry gate cannot be located at theinterface between Smc1/3 NBD domains andα-kleisin, because fusion of α-kleisin’s N- or C-terminal domains to their cognate NBDs doesnot prevent establishment of sister chromatidcohesion (43). To test whether the entry gateis instead situated at cohesin’s hinge, Smc1’sand Smc3’s hinge domains were replaced byp14 and MP1, respectively, which (like Smchinge domains) form tight pseudosymmetricheterodimers and whose N and C termini areclose enough to each other to permit formationof Smc coiled coils. Despite forming tripartiterings capable of ATP hydrolysis, these hinge-substituted Smc1/3 heterodimers neither asso-ciate with chromatin nor build sister chromatidcohesion (43). Cohesin’s hinge must there-fore have an essential function in addition todimerization.

It has proved possible to insert at short loopswithin Smc1’s and Smc3’s hinges amino acid



sequences encoding the human proteinsFKBP12 and FRB, which bind to each otherwith a low nanomolar KD only when FKBP12binds rapamycin. Remarkably, cohesin com-plexes with FKBP12 inserted into Smc3’s hingeand FRB inserted into Smc1’s hinge form sis-ter chromatid cohesion in the absence but notin the presence of rapamycin (43). Rapamycinblocks establishment of cohesion when addedbefore S phase but has no effect when addedimmediately afterward (43). These findings areconsistent with the notion that the hinge mustbe opened transiently during cohesion estab-lishment. However, this hypothesis is by nomeans proven, as clamping together of Smc1and Smc3 hinge domains may abolish cohesionestablishment for reasons other than prevent-ing hinge dissociation.

Smc1 and Smc3 hinge domains associatewith a low nanomolar KD, and ATP bindingand/or hydrolysis may be required to dissoci-ate them. The conundrum is that these NBDsare separated from the hinge by a 50-nm-longcoiled coil. This finding has led to the sugges-tion that the hinge is brought into direct prox-imity to the NBDs by folding the coiled coilseparating them.

Although passage of DNA double helicesthrough opened hinges may seem the obviousroute, it is not the only mechanism capableof trapping DNA. DNA trapping could alsoarise by passage through the hinge of a sec-tion of the cohesin ring itself (A. Leung, per-sonal communication). Consider, for example,the following scenario. If the DNAs destinedto be trapped were to lie orthogonally acrossthe parallel coiled-coil arms of an extended co-hesin ring and the coiled coils were now folded,bringing the hinge into contact with the NBDs,the DNAs would be trapped between sectionsof cohesin’s coiled coils, albeit in a tempo-rary embrace. If the hinge were to open, theNBDs were to disengage, and a central sectionof Scc1 α-kleisin polypeptide connecting theNBDs were to pass through the opened hinge,then subsequent hinge closure would create atwisted cohesin ring with the DNA trapped in-side the ring. Untwisting the ring, which would

be possible because the Scc1 linker is a singlepolypeptide, would transfer the change in link-ing number from the cohesin ring to the DNA,which would be wound around the ring. If bothsister DNAs were to lie side by side on cohesin’scoiled-coil arms at the initial step, then bothcould be trapped inside the ring by this process.

Arguments Against the Ring Model

A criticism of models postulating that cohesin’sassociation with chromatin is primarily topo-logical is that they cannot explain the ob-servation that cohesin is localized at specificsequences along the genome (65, 111). Al-though it is certainly true that DNA entrap-ment does not per se provide an explanationfor cohesin’s localization, entrapment does notpreclude sequence-specific localization. For ex-ample, rings that have trapped DNA may dif-fuse laterally along chromatin fibers until theymeet site-specific DNA-binding proteins (suchas CTCF; see section Does Cohesin Medi-ate Nonsister Connections Important for Tran-scriptional Control?) that bind to them with lowaffinity. Alternatively, localization could stemfrom the positioning of cohesin’s Scc2/4 load-ing factor at precise loci.

Some criticism has been directed specificallyat the strong ring model. There are a numberof circumstances in which cohesin persists onchromosomes even when sister chromatid co-hesion has been lost, and this is thought to beinconsistent with the notion that cohesin holdssister DNAs together by entrapping them in-side a single ring. One example is cohesin’sbehavior in eco1 mutants, where cohesin stillloads onto chromosomes but where sister chro-matid cohesion is not established during DNAreplication (112, 155, 180). A second exampleis the observation that sister chromatid cohe-sion of circular silent-mating type (HMR) locilooped out from yeast chromosomes by site-specific recombination is lost when silencingfactors are inactivated, and yet cohesin persistson the chromatin circles (12). These phenom-ena undoubtedly require explanations, but theyare not necessarily inconsistent with the ring



model. For example, Eco1 may be required toentrap both sister DNAs inside a single ring butnot for entrapment per se.

Other Models: The Handcuff Model

It has been postulated that cohesion is me-diated by interactions among separate co-hesin rings associated with each sister DNA(111). Weak ring models fall into this category(Figure 1b). Is there any evidence for inter-cohesin complex interactions? In yeast, whereit is easy to make diploid strains heterozygousfor differently tagged cohesin subunit alleles(each identically expressed at physiological lev-els from native promoters), little if any copre-cipitation is observed either with soluble co-hesin complexes or with complexes releasedfrom chromatin by nuclease digestion (50). In-deed, the former and latter have been shown tosediment similarly with velocities expected formonomeric complexes (199). Likewise, thereappears to be little or no FRET between YFP-and CFP-tagged versions of the same Smc sub-units in vivo in yeast (106). However, if co-hesin complexes that actually are engaged inholding sisters together were rare, then theymay have been missed by the above studies.More pertinent, therefore, is the finding thatchemical/translational cross-linking of the co-hesin ring’s three interfaces creates circular-ized rings that hold sister minichromosomestogether even after denaturation in 1% SDS athigh temperatures (49). This result should notbe possible if nontopological interactions be-tween different cohesin rings were an integralpart of the mechanism by which sister DNAs areheld together, because such interactions shouldbe destroyed by SDS treatment. It appears thatthe only way of salvaging nontopological weakring models would be to propose that minichro-mosome cohesion uses an atypical mechanism,which does not seem to be the case (143).

Soluble vertebrate cohesin complexes alsosediment with velocities expected of monomers(52, 100, 166). In contrast, it has recently beenreported that differently tagged α-kleisin sub-units coprecipitate, as do differently tagged

Smc1 or Smc3 subunits (212). A fundamen-tal problem with these experiments is that theproteins are overexpressed, which is known tofacilitate interactions between separate Smc1/3heterodimers. For example, separate yeastSmc1/3 heterodimers can be interconnected byScc1 when overexpressed in insect cells but notwhen expressed at physiological levels in yeastcells (50). Conclusions based on overexpres-sion studies should therefore be treated withgreat caution. Is there any evidence for inter-actions between identical cohesin subunits ex-pressed at physiological levels? Interestingly,mammalian tissue culture cells provide a nat-ural way of testing this hypothesis because theyexpress two variants of the Scc3 subunit, knownas SA1 and SA2. There is a consensus that thesetwo proteins cannot be coimmunoprecipitatedwith each other (52, 100, 166, 167, 212). Doesthis mean that cohesin does not form multi-mers? Not necessarily: It has been suggestedthat the α-kleisin subunits of two different tri-partite cohesin rings are connected by a singleScc3 molecule (Figure 2b) (212).

Do α-kleisin subunits associate with eachother without overproduction? In extracts fromstable cell lines expressing moderate levels ofthe Scc1 α-kleisin subunit tagged at its C termi-nus with myc epitopes, immunoprecipitation ofthe myc-tagged protein does not coprecipitatewild-type protein (194, 212). The argumentthat the C-terminal myc tag interferes withan all important kleisin-kleisin interaction ishard to reconcile with the finding that the verysame protein can sustain mouse development(K. Tachibana & K. Nasmyth, personal com-munication). The claim that different resultsare obtained in extracts from stable cell lines ex-pressing an N-terminally tagged α-kleisin sub-unit (212) also do not withstand careful scrutiny.Although it is expressed at a lower level than theendogenous protein, N-terminally tagged α-kleisin coprecipitates only very modest amountsof wild-type protein—indeed, no more than isimmunoprecipitated from extracts from cellsnot expressing a myc-tagged form (212). In con-clusion, there is currently little if any firm evi-dence that multimeric cohesin rings are formed



under physiological conditions, which does notnecessarily mean they do not exist.

Other Models: Bracelets

Another suggestion is that cohesin rings aremerely a storage form for soluble cohesin(65) and that sister chromatids are in factheld together by cohesin complexes that haveoligomerized. It is argued that the known inter-actions between cohesin ring subunits could beused to form not only rings but also oligomericfilaments or “bracelets” that somehow windaround sister chromatids (65, 121). This modelpostulates oligomeric cohesin complexes thathave hitherto defied detection and cannotexplain the chemical cross-linking experimentswith minichromosomes (49). Despite thisdrawback, the notion that Smc proteins mayform and function as multimeric filamentousstructures has received a boost from thesuggestion that MukB NBDs cannot engageone another while both are associated withMukF C-terminal domains (205). It has beenproposed that NBD engagement causes releaseof one end of symmetrical MukE4F2 hexamersfrom one of the two NBDs of MukB dimersand that the freed C-terminal MukF domaincan then interact with another MukB dimer ina process that could be reiterated to form fila-mentous structures. Whether this really occursunder physiological circumstances is unclear.

THE COHESIN RING CYCLE

Loading onto Chromatin

In animal and probably most fungal and plantcells, 90% or more of the cohesin boundto chromosomes dissociates during prophase(131). This process generates a large solublepool of mitotic cohesin complexes that arespared separase cleavage and reassociate withchromatin during telophase. Loading of co-hesin onto chromosomes is not confined topostmitotic or G1 cells. This is true not onlyin yeast, where α-kleisin expressed after Sphase associates with chromosomes (126, 186),

but also in HeLa cells, where cell lines sta-bly expressing green fluorescent protein (GFP)-tagged cohesin subunits have enabled measure-ment of chromosome residence times throughuse of inverse fluorescence recovery after pho-tobleaching (iFRAP). Such studies have re-vealed that most chromosomal cohesin has amean residence time of less than 25 min inboth G1 and G2 cells (35). G2 but not G1 cellspossess another population of cohesin (corre-sponding to one-third of the total), whose res-idence time is much longer and which maycorrespond to the cohesin pool actually en-gaged in holding sister chromatids together.Significantly, a large fraction of chromosomalcohesin cycles on and off chromatin severaltimes during the cell cycle. Cohesin engagedin holding sister centromeres together dur-ing mitosis turns over slowly if at all in yeast(209), consistent with the findings that non-cleavable α-kleisin subunits cannot form cohe-sion when expressed after DNA replication (51)and that wild-type Smc1/3 or α-kleisin subunitsexpressed in G2 cannot substitute temperaturesensitive variants (93, 163, 182).

Loading requires ATPase activity associatedwith Smc NBDs. Mutant Smc1 or Smc3proteins that can bind but not hydrolyze ATPdue to Walker B mutations form cohesin rings,but these fail to associate stably with chromo-somes (2, 3). The finding that engagement ofMukB’s NBDs is incompatible with both ofthem binding to the kleisin-like MukF protein(205) raises questions as to whether NBDengagement causes displacement of either theN- or the C-terminal domain of cohesin’sα-kleisin subunit and, if so, whether thisprocess is essential for stable association withchromatin. Pertinent to this issue is the fact thatcohesin complexes defective in hydrolyzingATP associated with either Smc1’s or Smc3’sNBD can be isolated as stable rings, implyingthat neither end of the α-kleisin subunit hasdisengaged from the NBDs. In addition, fulldisconnection of either NBD from its α-kleisinpartner is not necessary for establishing sisterchromatid cohesion, as yeast cells remain viablewhen Smc3’s C terminus is fused to α-kleisin’s



N terminus or when α-kleisin’s C terminus isfused to Smc1’s N terminus (43). It is thereforepossible but not proven that cohesin’s NBDsretain their association with both N- andC-terminal α-kleisin domains throughout theprocess of loading onto chromatin. Loadingalso involves cohesin’s hinge domain, whicheither acts as an entry gate (43) or conceiv-ably enables initial contact with chromatin asenvisioned for Bacillus subtilis Smc proteins (58).

Importantly, cohesin’s association withchromosomes requires several factors, some re-quired at all positions on the genome and othersonly at specific loci. The most important factoris the Scc2/4 complex. Association of cohesinwith chromatin at all sites within the genomedepends on this complex in Saccharomyces cere-visiae (14, 180), in Schizosaccharomyces pombe (7,178), in Xenopus extracts (36, 171), and in mam-malian cells (150, 197). As might therefore beexpected, mutations in Scc2 (110) or Scc4 (14)cause major defects in sister chromatid cohesion(110). It has been suggested that Mis4, Scc2’sS. pombe ortholog, may be part of an inde-pendent cohesion apparatus known as adherin(32). This possibility is unlikely because nei-ther Scc2 nor Scc4 is required to maintain sisterchromatid cohesion in postreplicative cells (7,14). Nevertheless, loading cohesin onto chro-mosomes may not be the sole function of theScc2/4 complex, as yeast scc2 mutants also havepartial defects in the loading onto chromo-somes of Smc5/6 complexes (94) and condensin(17)—defects that cannot be attributable tolack of cohesin loading. Scc2 is a large pro-tein containing multiple HEAT repeats withinits C-terminal domain (122, 128) and an Scc4-binding N-terminal extension whose lengthvaries enormously between species. Meanwhile,Scc4 is composed of TPR repeats (197). Thecomplex is highly conserved and is sometimesreferred to as the NIPBL/Mau2 complex in hu-mans (150).

In light of the cohesin-ring model, themain function of the Scc2/4 complex may beto promote entrapment of chromatin fibers bycohesin. How then might Scc2/4 perform this?It may act directly in the entrapment process,

helping to open the ring and/or bringing co-hesin into close proximity to chromatin fibers,or it may act only indirectly, modifying chro-matin fibers in a way that subsequently enablescohesin to entrap them. If Scc2/4 acts on co-hesin directly, the complexes must at some stageinteract physically with each other. Is there anyindication that this is the case? Scc2 and Scc4form a tight stoichiometric complex that forthe most part does not copurify with cohesinsubunits (7, 14, 197). However, small amountsof cohesin have been identified via mass spec-trometry in association with purified Scc2/4complexes (2), and Western blotting of cohesinin Scc2/4 immunoprecipitates or vice versa isconsistent with this finding (7, 170, 180).

There is accumulating evidence that co-hesin’s association with chromosomes requiresother factors in addition to the Scc2/4 com-plex. The best example has been discovered inextracts from Xenopus eggs, where associationwith chromatin of the Scc2/4 complex as wellas cohesin is dependent on the formation ofprereplication complexes (pre-RCs) (36, 171).Most Scc2/4 in these extracts is associated withthe Cdc7/Drf1 kinase (DDK), which is an es-sential component of pre-RCs (170). Consid-erable amounts of cohesin are also associatedwith Scc2/4. DDK and other pre-RC compo-nents such as Cdt1 are essential for loading bothScc2/4 and cohesin onto chromatin in theseextracts. The association between DDK andScc2/4 requires both the Cdc7 kinase subunit it-self and its regulatory Drf1 subunit and involvesScc2’s N-terminal domain bound by Scc4, butit does not require the C-terminal HEAT re-peat domain. Though not required for associa-tion with DDK, the latter is nevertheless essen-tial for cohesin’s loading onto chromatin. Theseobservations raise the possibility that DDK re-cruits Scc2/4 to pre-RCs prior to the initiationof DNA replication and that Scc2/4 in turn re-cruits cohesin. One suspects that once cohesinhas been recruited to the pre-RC by Scc2/4,the latter then facilitates chromatin fiber trap-ping both before and after the initiation ofreplication from the pre-RC. If this scenariois correct, then Scc2/4 has at least two key



functions: (a) to recruit cohesin to the vicinityof chromatin and (b) to catalyze chromatin fibertrapping.

Strangely, pre-RCs do not seem to be re-quired to recruit cohesin to yeast chromosomes(27, 186), and Scc2/4 complexes have not beenfound to be associated with pre-RCs. In addi-tion, expression of α-kleisin in G2 cells, whichno longer possess pre-RCs, leads to cohesin’sassociation with chromosomes with a patternthat is similar if not identical to that of α-kleisinloaded during S phase (93, 126). The findingthat a large fraction of cohesin in G2 HeLacells has a residence time of less than 30 min(35) also suggests that cohesin must frequentlyassociate with chromatin de novo long after pre-RCs have been disassembled. The linkage be-tween pre-RCs, recruitment of the Scc2/4 com-plex, and loading of cohesin onto chromatinmay therefore be specific to a short stage of thevertebrate cell cycle.

If, as the Xenopus studies suggest, Scc2/4interacts directly with cohesin prior to thelatter’s loading onto chromatin, the two com-plexes may not always maintain this connectionafter loading is completed. Chromosomespreads suggest that cohesin and Scc2/4 donot colocalize on yeast chromosomes (14).This notion has also been addressed throughmicroarray analysis of DNA associated withcohesin or Scc2/4 immunoprecipitates follow-ing formaldehyde cross-linking (ChIP-chip).Cohesin is enriched in a ∼40-kbp regionaround centromeres and at specific sites alongchromosome arms that frequently correspondto sites of convergent transcription (30, 39, 92).The distribution of Scc2/4 appears very differ-ent and resembles that of condensin and tran-scription factor IIIC associated with transferRNA (tRNA) genes (18). It has been suggestedthat cohesin first associates with loci whereScc2/4 is present and only then moves to sites ofconvergent transcription, having possibly beenswept there by RNA polymerase. Interestingly,a tRNA gene flanking the HMR locus hasbeen implicated in the establishment of sisterchromatid cohesion at silent mating-type genesat this locus, raising the possibility that cohesin

rings holding mating-type genes together maybe loaded at the neighboring tRNA gene (26).

A striking example of a specific locus in-volved in cohesin recruitment is the findingthat enrichment of cohesin for ∼40 kbp aroundS. cerevisiae centromeres depends on (a) the 150-bp CEN sequence (198), (b) specific proteinsthat associate with this sequence, such as thecentromere-specific histone H3, and (c) cen-tral kinetochore proteins such as Mtw1 (27).Remarkably, transfer of a CEN to a chromoso-mal arm is sufficient to enhance cohesin’s as-sociation with sequences in a 20–50-kbp inter-val surrounding the ectopic kinetochore (198).Cohesin’s concentration around centromereshas been visualized in live cells by imagingGFP-tagged Smc1 or Smc3 proteins (209). Theclearest images are those of metaphase cells inwhich Smc3-GFP forms a barrel-like structurealong the spindle axis, with kinetochores thathave bioriented and been split by spindle forcessituated at each end of the barrel and pole-to-pole microtubules running through the mid-dle of the barrel. Whether some of this co-hesin is associated with DNA sequences thathave been pulled apart by the spindle and areno longer cohesed (C loops) or whether mostof it is associated with neighboring pericentricDNAs that are still held together by cohesinis unclear. It has been suggested that cohesinon C loops may participate in intrachromatidcohesion, but there is no evidence for this in-teresting idea. How yeast kinetochore proteinspromote cohesin’s recruitment to pericentricchromatin is currently mysterious. One expla-nation for the ability of kinetochores to en-hance cohesin’s recruitment to a broad regionaround centromeres is that (together with theScc2/4 complex) they catalyze entrapment ofsequences close to the core centromere by co-hesin rings that subsequently diffuse (or evenare actively moved) to sequences 20 kbp or fur-ther away.

An instance of a factor implicated in cohesinrecruitment in S. pombe is the Swi6 HP1-likefactor, which binds to trimethylated K9 residueson histone H3 around centromeres and silentmating-type genes. Swi6 mutants have reduced



amounts of pericentric cohesin and defectivecentromeric sister chromatid cohesion (8, 125).It is uncertain whether HP1 orthologs have asimilar function in mammalian cells, where nei-ther their depletion through RNA interference(151) nor deletion of a pair of genes encodingthe histone H3 K9 methylase (82) appears toaffect centromeric cohesion adversely.

In summary, it is striking that work onXenopus extracts and budding yeast suggeststhat cohesin recruitment requires complexchromatin-bound machines in addition to theScc2/4 complex, namely pre-RCs, kineto-chores, and the tRNA transcription apparatus.Moreover, these factors may be only the tip ofthe iceberg. This possibility suggests that re-producing the recruitment process in vitro withdefined components, a step that will clearly beessential for any deep mechanistic understand-ing, will be a major challenge. Key questionsfor the future are whether chromatin fiber en-trapment inside cohesin rings is the mechanismby which they associate stably with chromatin,whether entrapment involves transient hingedissociation, whether and how the Scc2/4 com-plex participates in this process, and finally howchromosome locus–specific factors contribute.The invariable dependence of the loading pro-cess on these factors suggests that cohesin (andall other Smc complexes for that matter) showslittle or no promiscuity in its liaisons with chro-matin. One can only assume that such promis-cuity is avoided for good reasons, possibly toprevent establishing potentially damaging con-nections between chromosomal loci or jeop-ardizing efficient sister chromatid cohesion atcrucial regions such as around centromeres.

Establishment of Cohesion

Cohesin associates with chromosomes beforeDNA replication. What happens to cohesinrings associated with chromatin fibers ahead ofthe replication fork during and after its passage?Are they transferred in cis to sister chromatidsimmediately after the fork in a manner thatsometimes leads to sister chromatid cohesion,or are they ejected from the chromosome?

According to one particular version of thestrong ring model, coentrapment of sisterDNAs may be generated from passage of repli-cation forks through rings that had previouslyentrapped the unreplicated ancestral fiber (50).The finding that temperature-sensitive (ts)scc2-4 yeast cells can establish sister chromatidcohesion (at the URA3 locus) at the restrictivetemperature after release from arrest in earlyS phase by hydroxyurea at the permissive tem-perature has led to the conclusion that “sisterchromatid cohesion can be built exclusivelywith cohesin that was already bound to chro-mosomes before arrival of the replication fork”(93, p. 789). The problem is that considerablereplication takes place in cells arrested for longperiods in hydroxyurea, and it is possible thatthe cohesion measured had been producedduring the arrest. More rigorous experimentswill be required to settle this key issue. More-over, the recent finding that cohesion can beestablished on fully replicated chromosomes inthe apparent absence of replication forks (161,163, 190) implies that replication throughcohesin rings cannot be the only mechanism(if at all) by which cohesion is generated.

What is much clearer is that when expressedin G2 or M phase yeast cells, componentsof the ring complex—namely Smc1/3 and α-kleisin subunits—associate with chromosomesbut, under normal circumstances, fail to estab-lish cohesion even in the presence of preexist-ing sister chromatid cohesion (51, 93, 161, 163,190). A corollary is that there can be little orno turnover of these cohesin subunits withinthe cohesive structures holding sisters together.What then enables cohesin present during Sphase to establish cohesion but not that presentin G2 or M phase? A clue lies in genetic studiesthat have revealed genes required for establish-ing cohesion but not for loading it onto chro-mosomes or for maintaining cohesion alreadyestablished. Important but not essential are avariety of proteins thought to travel with repli-cation forks (73, 207) that are either implicatedin DNA replication, such as Ctf4 (33), or inthe loading or unloading of proliferating cellnuclear antigen (PCNA) onto chromosomes



such as the RFCCtf18/Dcc1/Ctf8 complex (104,105).

More crucial is an acetyl transferase knownas Eco1, whose acetylation of a pair of lysines(K112 and K113) within Smc3’s NBD, close tobut not immediately adjacent to its ATP bind-ing pocket (Figure 1c), is essential for cohesionestablishment (5, 143, 189, 211). Orthologs ofEco1 are encoded by most but not all eukaryoticgenomes (121), and mutations invariably resultin cohesion defects (176, 201). Humans con-tain two such proteins, Esco1 and Esco2 (64).Mutations in the gene encoding Esco2 are re-sponsible for the developmental defects associ-ated with Roberts syndrome and SCphocomelia(149, 192), and cells from these patients are de-fective in centromeric cohesion. Furthermore,the Smc3 residues corresponding to K112 andK113 are highly conserved among eukaryotesand are acetylated in human tissue culture cells(211). In yeast, Smc3 acetylation increases dur-ing S phase in a manner that appears to depend(a) on the Scc2/4 complex (189), suggesting thatit only occurs if cohesin can associate with chro-matin, and (b) at least partly on the prereplica-tion protein Cdc6 (5), implying at least partialdependency on replication forks. Smc3 acety-lation remains high for much of the G2 andM phases, decreasing around the time cells un-dergo anaphase (5). This finding suggests thatdeacetylation may be triggered by cohesin’s dis-sociation from chromatin.

What effect does acetylation of K112 andK113 have on cohesin rings that enables themto establish connections between sister DNAs?The discovery that inactivation of Pds5 inS. pombe (a nonessential cohesin subunit in thisorganism) suppresses lethality caused by dele-tion of Eco1’s ortholog Eso1 (172) raised thepossibility that Eco1’s modification of cohesincounteracts aspects of its function that oth-erwise interfere with cohesion establishmentduring S phase. Without this “antiestablish-ment” activity, acetylation of Smc3 is at leastpartially redundant. Subsequent genetic stud-ies in budding yeast have revealed that nullalleles of Rad61/Wapl and point mutationswithin specific domains of essential cohesin

subunits—namely Smc3, Pds5, and Scc3—havea similar effect, enabling proliferation withoutEco1 (5, 143, 169). It has been suggested thatalthough Eco1 itself acts during DNA repli-cation, Smc3 acetylation might be requiredpostreplication to counteract a tendency ofRad61 to remove cohesin from chromosomes.Contrary to the predictions of this model,Rad61 does not reduce cohesin’s associationwith chromosomes in S. cerevisiae, but ratherpromotes it (143, 169, 195). Moreover, there islittle or no evidence that cohesin’s associationwith chromatin in postreplicative cells is greatlydestabilized by a lack of Smc3 acetylation (143).

Despite the very different phenotypescaused by inactivating Rad61 in yeast andWapl in HeLa cells (see section DissociationDuring Prophase and Anaphase, below), itis nevertheless likely that these orthologsoperate using similar mechanisms. Bothform stable stoichiometric complexes withPds5 (34, 88, 143). Rad61 (at least) alsobinds Scc3, whose phosphorylation dur-ing mitosis in mammalian cells promotescohesin’s dissociation from chromosomesduring prophase (52). Whether these proteinspromote or reduce cohesin’s association withchromosomes may depend on their state ofmodification and the stage of the cell cycleas well as any evolutionary divergence pro-ducing different properties. The Smc3 NBDappears to be the target of antiestablishmentactivities and its acetylation presumably servesto counteract these during S phase. However,the process normally mediated by acetylationof K112 and K113 can also be achieved,albeit less efficiently, by a variety of mutationsclose to Smc3’s ATP binding pocket (5, 143),including those that do not mimic acetylation.In summary, these studies indicate that co-hesin establishment requires a reorganizationof Smc3’s NBD that can be hindered byRad61/Wapl, Pds5, and Scc3 proteins and thatpart of the function of Smc3’s acetylation byEco1 is to counteract this inhibition.

The finding that Smc3 acetylation is at leastpartly dependent on DNA replication (5) sug-gests that a lack of Smc3 acetylation could



explain why cohesin subunits produced in G2or M phase cannot generate sister chromatidcohesion despite forming cohesin rings that as-sociate stably with chromatin. Consistent withthis notion is the finding that overexpression ofEco1 enables (some) postreplicative cohesionestablishment (190). Remarkably, DNA dam-age also enables cohesin to establish sister chro-matid cohesion during M phase, even on chro-mosomes that have not been damaged (161,190), raising the possibility that signals stem-ming from local DNA damage and involvingphosphorylation of cohesin’s α-kleisin subunitby the Chk1 kinase (54) somehow stimulatemore widespread acetylation of cohesin sub-units outside of S phase (55).

How might the act of DNA replication orsignals stemming from replication forks facil-itate Smc3 acetylation by Eco1? A clue is thefinding that Eco1’s N terminus binds PCNA,at least in vitro, and that point mutations ina QXXL/I motif abolish binding and causeinviability (114). The notion that Eco1 may berecruited to replication forks through bindingto PCNA would explain why mutations inthe RFCCtf18/Dcc1/Ctf8 complex compromisecohesion more than DNA replication if this al-ternative polymerase clamp loader is especiallyimportant in loading those PCNA clamps thatrecruit Eco1 to replication forks. Whether theplethora of genes thought to regulate DNAreplication and to promote sister chromatidcohesion, e.g., Ctf4, Tof1/Csm3, or Chl1,facilitate cohesion establishment by helping torecruit Eco1 to replication forks should be easyto test. However, whether Eco1 is indeed re-cruited to replication forks (93) is far from clear.

An important feature of cohesion estab-lished in postreplicative cells is that it dependsnot only on de novo Eco1 activity but also onpreexisting cohesion (161, 163, 190); it cannotbe generated if the sisters are not already paired.This is presumably why cohesion must be es-tablished during DNA replication (186), evenif it can in principle be repaired or reinforcedby new cohesion produced in G2- or M-phasecells. The ability to generate new cohesin link-ages after S phase may be particularly important

in cells that spend very long periods in G2, forexample oocytes where linkages produced dur-ing S phase may decay with age. Does cohesionregeneration actually occur under normal cir-cumstances? The observation that the meiosis-specific Smc1 isoform (Smc1B) is synthesizedafter S phase and yet contributes to sister chro-matid cohesion, albeit so far only in cells arti-ficially induced to enter M phase, suggests so(138).

Dissociation During Prophaseand Anaphase

Although the mechanics of cohesin’s removalfrom chromosomes may be simpler than theestablishment of cohesion in the first place,the process must nevertheless be very tightlyregulated. Chromosome biorientation duringmitosis is impossible without sister chromatidcohesion, and its premature loss would bedisastrous. In most eukaryotic cells, cohesin’sdissociation takes place during two phases ofmitosis (98, 131, 166, 194). The first (knownas the prophase pathway) takes place duringprophase and prometaphase, when most butnot all cohesin dissociates from chromosomearms but not from centromeres. The sec-ond takes place shortly before the onset ofanaphase, when all remaining cohesin (mainlyat centromeres but also on arms) dissociatesdue to cleavage of its α-kleisin subunit byseparase (53, 89, 118, 185, 187, 202). Thesetwo processes have different mechanisms inaddition to different temporal regulation, as theprophase pathway does not involve cleavage byseparase (166). The prophase pathway may notbe an essential aspect of mitotic chromosomesegregation and is largely if not completelyabsent during yeast mitosis. Moreover, little orno cohesin dissociates from chromosome armsin prometaphase during meiosis I, when sisterchromatid cohesion along chromosome armsis essential for holding bivalent chromosomestogether. Bivalents are eventually convertedto dyad chromosomes exclusively (possibly)due to cleavage of arm cohesin complexes byseparase (10, 79, 86, 87, 175).



How cohesin is removed from chromosomearms by the prophase pathway is not known.The ring model predicts that it must involvering opening, but whether (and if so how) thistakes place is not known. Key questions arewhether exit and entry gates are the same andwhether they both involve cohesin’s hinge.The process is facilitated by the Aurora Band polo-like kinases (PLK) (91, 99, 167),by PLK-mediated phosphorylation of theC-terminal domain of cohesin’s SA1/2 Scc3subunits (52), and by condensin I (60). AuroraB’s role may be quite indirect, either ensuringthat Sgo1, a protein that protects cohesinfrom the prophase pathway, is directed tocentromeres, and/or promoting recruitmentof condensin I to chromosome arms (95).However, these effectors/processes have quitemodest roles, as most cohesin still dissociatesfrom chromosome arms in their absence. TheWapl protein is more crucial. Its depletionfrom mammalian tissue culture cells, eitherby RNA interference or by gene inactivation( J.-M. Peters, personal communication),causes most cohesin to remain on chromosomearms (34, 88). Wapl is found associated withcohesin, forms a particularly stable complexwith its Pds5 subunit, is hyperphosphorylatedduring mitosis (as are α-kleisin, Pds5, and Scc3SA1/2), and contributes to turnover of cohesinon interphase chromosomes. Importantly,Wapl’s function is not merely to promotehyperphosphorylation of SA2. The Scc2/4complex also dissociates from chromosomesduring prophase, which may also contribute tothe removal of arm cohesin (197).

What is the function of the prophase path-way? Unlike the APC/C-separase pathway, itmay not be a universal feature of chromo-some segregation. There is no intrinsic rea-son why α-kleisin cleavage could not deal withall cohesin (88). Moreover, the prophase path-way has not yet been implicated in any ob-vious regulatory function. Nevertheless, re-moval of cohesin from chromosome armsmay facilitate their deconcatenation by TopoII during prometaphase and metaphase andmay thereby contribute to timely disjunction

at anaphase. Another potential function israised by the observation that the large solublepool of cohesin created by the prophase path-way is not cleaved by separase at the metaphase-to-anaphase transition (168, 194). A large frac-tion of this pool reassociates with chromosomesduring telophase and very possibly has impor-tant functions regulating transcription and thestructure of chromatin during interphase (131).If most cohesin were removed from chromo-somes by separase, then reassociation wouldhave to await resynthesis of its α-kleisin sub-unit, as occurs in budding yeast, with potentiallygrave consequences for transcriptional control.

The prophase pathway must not removeall cohesin from chromosomes, as doing sowould compromise their biorientation on mi-totic spindles. Indeed, centromeric cohesin isprotected from the prophase pathway due tothe centromere-specific Sgo1 protein (77, 108,146). Sgo1 is a member of a class of pro-teins known as shugoshins, whose foundingmember is the Mei-S332 protein in Drosophilamelanogaster (75). Shugoshins possess a con-served coiled-coil domain that binds the ABCPP2A holoenzyme (139, 174), which is alsolocalized to centromeres in mitotic cells andmay therefore counteract phosphorylation ofcohesin subunits, Wapl, and Scc2/4. Whethershugoshins protect centromeric cohesin in mi-tosis by recruiting PP2A is still unclear, andthere are conflicting reports as to whetherPP2A’s recruitment to centromeres requiresshugoshins (140) or vice versa (80, 174).

Cleavage by Separase

The metaphase-to-anaphase transition is thepoint of no return. There is no going backonce cells destroy all sister chromatid cohe-sion (186). There is overwhelming evidence inyeast that sister chromatid disjunction is trig-gered by cleavage of cohesin’s α-kleisin subunitScc1 by separase (178, 185, 187). Likewise, res-olution of chiasmata, which converts bivalentchromosomes to dyads at the first meiotic divi-sion and requires loss of cohesion along chro-mosome arms, is achieved by cleavage of the



meiosis-specific α-kleisin Rec8 (10, 79). Sep-arase activity is also required in Caenorhabdi-tis elegans (153), in Drosophila melanogaster (71),in mammals (87, 89, 202), and in Arabidopsis(96). Whether α-kleisin cleavage is either nec-essary or sufficient in eukaryotic cells other thanyeast is less clear cut. A noncleavable version ofScc1 interferes with sister chromatid disjunc-tion in HeLa cells, although it may not com-pletely block loss of centromeric cohesion (53),whereas a supposedly noncleavable version ofRec8 merely slows down the process of chi-asmata resolution (86). The latter causes thefirst meiotic division to be aborted and causessterility in spermatocytes, but not in oocytes.The finding that inactivation of separase pro-tease activity completely blocks Rec8’s removalfrom bivalents (87), whereas mutation of cleav-age sites (detected in vitro) has only a partial ef-fect (86), implies that the failure to arrest com-pletely sister chromatid disjunction is caused bycryptic cleavage sites that are not detected byin vitro assays and are still present in suppos-edly noncleavable variants. There is thereforeno overwhelming reason to doubt that α-kleisincleavage is both necessary and sufficient for sis-ter chromatid disjunction in most if not all eu-karyotic organisms, even if rigorous proof forthis assertion is still lacking. Thus, there is nojustification at present for concluding that “thecohesin cleavage model does not ring true” (45).

Separase activity must be highly regulatedand is inhibited for much of the cell cyclethrough its association with a specific inhibitorychaperone known as securin (187). During mi-tosis, phosphorylation of separase (on serine1121) by Cdk1 (159) induces stable bindingof cyclin B/Cdk1 (42). This also inactivatesthe protease, a process especially important inpostmigratory primordial germ cells (67) andpreimplantation mouse embryos (66), in whichsecurin levels may be lower than in other cells.Separase is only (fully) activated when all chro-mosomes have bioriented, which liberates theAPC/C’s Cdc20 activator protein from seques-tration by its Mad2 inhibitor (116, 119). Thispermits Cdc20 to recruit securin and cyclin Bto the APC/C, leading to their ubiquitinylation

and subsequent proteolysis. Upon libera-tion from securin and cyclin, active separasecleaves the Scc1 subunits of cohesin (53, 185)still associated with chromosomes (168, 194),which induces cohesin’s dissociation from chro-matin and triggers sister chromatid disjunction.Though separase normally only removes co-hesin that has resisted the prophase pathwayfrom chromosomes, it appears capable of re-moving larger amounts at the metaphase-to-anaphase transition, for example when Wapldepletion inactivates the prophase pathway(88). Crucially, it is the APC/C-separase path-way, and not the prophase pathway, that is reg-ulated by the SAC (107). Due to its inhibi-tion by two different APC/C substrates (se-curin and cyclin B) whose ubiquitinylation isblocked by Mad2, separase can only be fullyactivated once the SAC has been turned off.Some suggest that separase may be partially ac-tive during prometaphase, at least when cellsare arrested for prolonged periods in a mi-totic state by spindle poisons such as noco-dazole (118). How separase selectively cleavesonly α-kleisins associated with chromatin is notunderstood (168). Separase is a huge protein(with over 2000 residues in humans) with anN-terminal domain containing 26 Armadillo(ARM) repeats separated from a pair of caspase-like protease domains (only one of which is ac-tive) by an unstructured central region. Securinis thought to bind to the N-terminal ARM re-peats, but it may also interact with the pro-tease domain (62). It is conceivable that sep-arase’s ARM-repeat-containing domain bindschromatin and thereby juxtaposes the proteasewith its α-kleisin substrate (193).

One of the more remarkable phenomenaconcerning sister chromatid disjunction is themechanism that protects Rec8 α-kleisin in thevicinity of centromeres from separase cleav-age at the first meiotic division (134). Cleav-age along chromosome arms is essential for theresolution of chiasmata at meiosis I, but it mustnot occur at centromeres because their cohe-sion is essential for biorientation of dyad chro-mosomes at meiosis II. It has long been knownthat the persistence of centromeric cohesion



after meiosis I in D. melanogaster depends on acentromeric protein termed Mei-S332 (40, 75).Identification of fungal orthologs (78, 102, 136)revealed a family of conserved proteins (nowknown as shugoshins, described above) thatare associated with AB’C PP2A holoenzymesduring meiosis (139). Humans possess twomembers of this family, Sgo1, which protectscohesin from the prophase pathway (108, 146),and Sgo2, which protects centromeric cohesinfrom separase during meiosis I (97). Shugoshinscontain an N-terminal homodimeric parallelcoiled coil whose docking onto PP2A’s C and B′

subunits recruits it to centromeres and protectsRec8 from separase (208). How does recruit-ment of PP2A to centromeres block α-kleisincleavage at this location? If phosphorylation,either of separase or of cohesin, were neces-sary for Rec8 cleavage, then PP2A might actby removing the requisite phosphate groups.The finding that Rec8’s mitotic counterpartScc1 cannot be protected by shugoshin/PP2Acomplexes suggests that PP2A’s target is Rec8itself (181). Rec8 is indeed phosphorylated atmultiple serines and threonines during meiosisI (9). Crucially, substitution of these residuesby alanine greatly reduces cleavage and hin-ders chiasmata resolution, whereas substitu-tion with aspartate, mimicking phosphoryla-tion, causes precocious loss of sister centromerecohesion (V. Katis & K. Nasmyth, unpub-lished results). The notion that shugoshinsprotect centromeric cohesion by dephosphory-lating Rec8 is consistent with the finding thatRec8 phosphorylation is necessary for its effi-cient cleavage by separase in vitro (86).

A Case Against Cohesin?

In the light of the above discussion, it is curi-ous that doubts have recently been raised as towhether cohesin really holds sister centromerestogether up until the onset of anaphase andwhether this linkage is destroyed by theAPC/C-separase pathway (15, 23, 38, 45). Ithas been suggested that neither APC/C (38) norseparase (37, 45) is required for the disjunctionof centromeres at anaphase, that depletion of

cohesin does not prevent chromosome biorien-tation (23), that the fraction of Scc1 moleculescleaved by separase is insufficient to explain sis-ter chromatid disjunction at anaphase, and thatexpression of noncleavable Scc1 in mammaliancells affects disjunction of sister chromatidarms more severely than that of centromeres(23, 127).

The first two of these claims are basedlargely on the results of knocking down APC/Csubunits or separase by RNA interference.Both sets of proteins are notoriously difficultto deplete efficiently and rapidly using thistechnique. More importantly, these claims areclearly inconsistent with a wealth of data onthe effect of mutations in a wide variety ofmicro-organisms and invertebrates. One wouldhave to argue that sister chromatid disjunctionis mediated by a very different mechanism invertebrate cells. However, even this claim isnot borne out by the facts. Complete depletionof the APC/C using gene deletion in hepato-cytes induced to proliferate by partial hepatec-tomy causes them to arrest in metaphase (203),whereas mouse embryonic fibroblasts lackingseparase, again due to gene deletion, clearly failto disjoin sister chromatids upon activation ofthe APC/C. In addition, depletion of separasein oocytes, via gene deletion, prevents the con-version of bivalent chromosomes into dyads atmeiosis I, and this failure is accompanied bythe persistence of cohesin along their interchro-matid axes (87).

The claim that at least some degree ofchromosome biorientation can take place inmitotic cells lacking cohesin is less controver-sial. Some studies report considerable biorien-tation in cohesin-depleted cells (74, 130, 157),whereas others describe major defects (173,183). This is an area in which the limitationsof RNA interference are fully exposed. It is dif-ficult to exclude the possibility that apparentlynormal biorientation is caused by incompleteprotein depletion, and it can be argued thatmore severe defects may be due to off-targeteffects. In contrast, there is a consensus that co-hesin inactivation via ts mutations, Scc1 cleav-age, or gene repression invariably triggers the



SAC and causes sister chromatid disjunction inthe presence of high levels of cyclin B (130, 183)or securin (110). What about the argument thatinsufficient Scc1 is cleaved to explain anaphase(23, 45)? The concentration of cleavage frag-ments is indeed low compared to intact pro-tein, but there is a very good reason for this: therapid degradation of the cleavage fragments bythe N-end rule Ubr1 ubiquitin protein ligase(137).

The last argument is that despite major de-fects in disjoining sister chromatids at anaphase(53), cells expressing noncleavable Scc1 proteinappear to separate sister centromeres (23). Itis difficult to ascertain the exact importance ofcleavage without observing mitoses in live cellsthat had expressed physiological levels of non-cleavable protein prior to the preceding S phase.This has not yet proved possible. It is moreoverdifficult to be certain that the noncleavable vari-ants used hitherto are truly noncleavable anddo not contain cryptic cleavage sites. Despitethis drawback, a case can be made that evenif the noncleavable proteins are not fully non-cleavable and even if they are not expressed atphysiological levels, they should surely disruptdisjunction of sister centromeres as severelyif not more severely as that of chromosomearms, which appears not to be the case. Thisis not a strong argument, but it does suggestthat the role of cohesin cleavage merits furtherinvestigation.

Despite the the fact that the case againstcohesin is weak, might “Topo II and cohesincontribute equally to regulate sister chromatidassociation” (183, p. 2301)? There is no ques-tion that decatenation of sister DNAs by TopoII is required for sister chromatid disjunction,and it is certainly possible that sister DNAintertwining may help cells lacking cohesinto biorient chromosomes for a limited periodduring the early phases of mitosis. However,such cells do not exist in nature as far as weknow. Moreover, there is no evidence that TopoII is ever inhibited sufficiently during mito-sis to ensure that DNA concatenation can re-sist spindle forces. It therefore makes littlesense to regard the DNA concatenation/Topo

II system as a backup to the cohesin/separasesystem.

There is one clear, albeit peculiar, exampleof cohesion between sister chromatid DNAsthat is both cell-cycle regulated and indepen-dent of cohesin, namely sister chromatid cohe-sion between tandem rDNA repeats on chro-mosome XII in yeast. Possibly due to their highrates of transcription, sister rDNA loci in yeastare entangled via a poorly understood cohesin-independent mechanism (11, 19, 165). Artificialcleavage of cohesin’s Scc1 subunit by TEV pro-tease therefore triggers disjunction of most sis-ter chromatids but not of sister rDNA repeats.A different mechanism, possibly DNA decat-enation, must trigger sister rDNA disjunction.rDNA loci do not disjoin particularly slowly inanimal cells, and it is unclear whether the pro-cess described in yeast also operates in animalcells.

ROLES BEYOND COHESION

Cohesin and Double-Strand-BreakRepair

Cohesin plays key roles in the repair of DNAdouble-strand breaks in mitotic (17, 154) andmeiotic (29, 81, 191) cells. In mitotic cells, twodifferent populations of cohesin contribute tothe repair process: cohesin engaged in holdingsisters together at the time of the break and co-hesin subsequently recruited to chromatin sur-rounding the break itself (162, 188). Cohesin isparticularly important in meiotic cells, whereprogrammed double-strand-break repair cre-ates the reciprocal recombination events (chi-asmata) that hold bivalent chromosomes to-gether (134). One of the remarkable features ofthe meiotic process is that double-strand breaksare repaired preferentially using nonsister chro-matids, a property possibly facilitated by replac-ing mitotic α-kleisin subunits (Scc1/Rad21) bymeiosis-specific versions (Rec8) (4, 206). Mam-mals also express meiosis-specific versions ofSmc1 (Smc1B) (138) and Scc3 (STAG3) (135)and there are therefore a large variety of co-hesin complexes during spermatogenesis and



oogenesis. Cohesin forms the backbone of thesynaptonemal complex (28, 81) and presumablyplays fundamental roles in orchestrating therepair of meiotic double-strand breaks (160).Whether cohesin is actively recruited to thesites of double-strand breaks during meiosis asit is in mitotic cells is not known. Because of itscrucial role during recombination, loss of func-tion usually leads to a checkpoint-induced ar-rest at the pachytene stage (4, 138), which ham-pers analysis of its roles and regulation duringsubsequent chromosome segregation (9).

Cohesin and Mono-orientationDuring Meiosis I

Work on S. pombe and Arabidopsis indicates thatcohesin is involved in another meiosis-specificfunction, namely mono-orientation of sisterkinetochores during meiosis I. During mito-sis, microtubules pull sister kinetochores inopposite directions (biorientation), but duringmeiosis I they instead pull maternal and pa-ternal kinetochore pairs in opposite directions.Electron micrographs show that sister kineto-chores are seamlessly bound together in a singlestructure at this stage (41). Meiosis-specific“monopolin” proteins confer this property(103, 133, 181, 210), but how do they function?In both S. pombe and Arabidopsis, mutations inthe meiosis-specific α-kleisin Rec8 lead to bior-ientation of sister kinetochores (13, 196) as wellas to sister chromatid cohesion defects, raisingthe possibility that cohesin may connect sisterkinetochores during meiosis I in a way that pre-cludes their biorientation. It appears that Rec8,but not its mitotic equivalent Rad21, managesto create sufficient cohesion between sisterkinetochores to preclude their biorientation(145), a process that only occurs in meiotic cellsand is regulated by monopolins. If such a pro-cess is also responsible for mono-orientation inS. cerevisiae, which is not known, then it is notan exclusive property of Rec8-containing co-hesin complexes, as cells expressing the mitoticScc1 instead of Rec8 can also mono-orienttheir meiosis I kinetochores (181).

Does Cohesin Mediate NonsisterConnections Important forTranscriptional Control?

The first indication that proteins involved insister chromatid cohesion may have functions inaddition to holding sister chromatids togetherwas the description of Roberts syndrome (141)and SCphocomelia (56) in humans. These arerare autosomal recessive conditions associatedwith limb and growth deficiencies, craniofacialanomalies, and mild to severe mental deficiency.Mitotic chromosomes from these patients arecharacterized by heterochromatin repulsion,in particular at centromeres, indicating pre-mature (albeit partial) loss of sister chromatidcohesion (31, 72, 176). Due possibly to theirdefective sister chromatid cohesion, culturedRoberts syndrome cells have mitotic defects(177), but it is unclear whether these defectsalone account for either the severe (in the caseof Roberts) or the mild (in the case of SCpho-comelia) developmental defects. The presenceof severely and mildly affected individuals inthe same sibship (children from the same setof parents) indicated that the two disordersare allelic, and it is now clear that both arecaused by mutations in ESCO2 that abolish itsfunction (149, 192). What is unclear is whetherthe variable developmental defects are causedby variations between individuals in how theirembryonic cells react to partial loss of sisterchromatid cohesion. Affected cells may differin the extent of their arrest by the SAC or intheir propensity to undergo apopotosis. How-ever, given the superficial similarity of the limbdefects with those caused by known regulatorsof gene expression, for example HOX genes, itis not inconceivable that the highly pleiotropicdevelopmental defects arise due to defects inthe expression of genes regulated by Esco2through its acetylation of cohesin subunits.SCphocomelia birth defects are similar to thosecaused by thalidomide, which conceivably mayact by interfering with processes regulated bycohesin.

A stronger clue that cohesin may have non-mitotic functions stems from the discovery that



half or more patients with Cornelia de Langesyndrome (CdLS) (20) have mutations in onlyone allele of the gene encoding human Scc2,known also as Nipbl due to its homology withthe fly ortholog Nipped-B (85, 179). CdLSis a dominantly inherited multisystem devel-opmental disorder characterized by growthand cognitive retardation; abnormalities of theupper limbs; gastroesophageal dysfunction;cardiac, ophthalmologic, and genitourinaryanomalies; hirsutism; and characteristic facialfeatures. These and other defects are causedby only modest (30%) reductions in the levelof Scc2/Nipbl protein, which does not appearto be accompanied by major defects in sisterchromatid cohesion. The homology with Scc2raised the possibility that CdLS defects maybe caused by reduced cohesin loading, a notionconsistent with the subsequent discovery thatmilder forms of CdLS are caused by Smc1Aand Smc3 mutations (21, 117). Consistent withthe notion that CdLS is caused by misregu-lated gene expression, the D. melanogaster Scc2ortholog, Nipped-B, facilitates long-rangeenhancer-promotor interactions, at least forcertain genes whose regulatory sequenceshave been mutated (25, 142). Furthermore,mutations in mau-2, the C. elegans Scc4 or-tholog, cause defects in axon guidance (6) andtwo cohesin subunits, Scc1/Rad21 and Smc3and have been implicated in expression ofthe hematopoietic transcription factor Runx1in zebrafish (63). Despite these findings, thepossibility that developmental cohesinopathiesare caused by knock-on effects of compromis-ing sister chromatid cohesion cannot be fullyexcluded.

The discovery that cohesin resides withinthe nuclei of most postmitotic cells, includ-ing neurons (200), and that cohesin inacti-vation causes pruning defects in postmitoticmushroom-body γ-neurons in Drosophila, duein part to a lack of expression of the ecdysonereceptor, demonstrate unequivocally that co-hesin does indeed have nonmitotic functions,including the regulation of transcription (130,148). In yeast, such functions include restricting

the spread of silencing at silent mating-typeloci (24) and regulating transcriptional termi-nation (47). It is striking that in mammaliancells cohesin frequently colocalizes with thesite-specific zinc finger DNA binding proteinCTCF, which has been proposed to defineboundaries between active and inactive chro-matin domains (129, 144, 158, 200). CTCFappears necessary for cohesin’s localization toCTCF sites but not vice versa, suggesting thatCTCF recruits cohesin and not the other wayaround (144, 200). Recruitment in this sensedoes not necessarily mean that CTCF (togetherwith the Scc2/4 complex) actively promotescohesin’s loading onto chromosomes. Indeed,the amount of cohesin associated with chro-matin is little changed in cells depleted forCTCF (200). If cohesin’s loading onto chro-matin invariably involves entrapment of DNAby cohesin rings that can subsequently dif-fuse laterally, then CTCF may merely deter-mine where on chromosomes entrapped ringsaccumulate.

At the imprinted H19/IGF2 locus whereCTCF and cohesin are recruited in an allelespecific manner to a site between the two genes,both proteins have a role in preventing acti-vation of the maternal IGF2 allele by an en-hancer at the 3′ end of the H19 gene (200).The ability of cohesin rings to entrap sisterchromatin fibers (49) raises the possibility thatthey may also be capable of entrapping dis-tant chromatin segments (within a single ring),thereby creating loops. Cohesin may there-fore be a key regulator of long range interac-tions between enhancers and promoters. Cross-linking studies indicate that such loops formin a cohesin-dependent fashion at the IFNGlocus in T cells. If so, they do not seem cru-cial for transcriptional induction, as this is notgreatly affected by cohesin depletion (48). Thefinding that cohesin is recruited to the CTCFsites of V and J segments of immunoglobulingenes in pro B cells raises the possibility thatcohesin may contribute to contraction of thelocus thought to facilitate gene rearrangement(22).



It therefore seems very likely that thedevelopmental defects caused by a modest dropin the level of the Scc2/4 (Nipbl) complex aredue to changes in the loading of cohesin ontochromosomes and thereby to changes in geneexpression. To explain the devastating effect ofsuch a small change in a single factor, it may benecessary to postulate that different genes/lociare in constant competition for a limitingamount of Scc2/4 and that only a small dropin its abundance causes major changes in theamount of cohesin recruited to particular loci.

Microarray studies suggest that cohesin fre-quently colocalizes with RNA polymerase inDrosophila tissue culture cells (113). This raisesthe possibility that cohesin may be a “hitch-hiking device” to which other factors can bind

while it (cohesin) moves along chromosomeswith RNA polymerase.

A Function at Centrosomes?

Two studies have recently detected cohesin sub-units at spindle poles (204) or centrosomes (83).Whether or not this population of cohesin hasan important role at these locations is less clear.Cohesin depletion does indeed cause spindlepole defects, but it is difficult at this stage to ex-clude the possibility that these are indirect con-sequences of prior mitotic defects caused by thelack of sister chromatid cohesion. Nevertheless,these are intriguing findings in the light of thesuggestion that separase may have a function incentriole disengagement (184).

FUTURE ISSUES

1. Does cohesin’s stable association with chromatin involve trapping of chromatin fibers?Are sister DNAs held together due to their entrapment inside monomeric cohesin rings?

2. Is transient dissociation of cohesin’s hinge required for DNA entrapment? Does theScc2/4 complex facilitate DNA trapping?

3. What are the mechanistic consequences of Smc3 acetylation?

4. What are the structural consequences of ATP binding and hydrolysis by Smc proteins?

5. Is postreplicative cohesion establishment on undamaged chromosomes of physiologicalimportance?

6. Does cohesin regulate transcription by generating loops between distant DNAs and doEsco1/2 modulate cohesin activity for the purposes of transcriptional control?

7. To answer these questions, it will be important to (a) determine atomic structures ofthe Smc3 NBD-kleisin interface, the non-Smc proteins Scc3, Scc2/4, Wapl, and Pds5;(b) develop in vitro systems that recapitulate key steps in cohesin’s association with chro-matin fibers; and (c) develop assays to measure cohesion and/or DNA loop formation invitro in systems other than budding yeast.

DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings thatmight be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

We thank Arthur C. Leung for pointing out the possibility that Scc1 passage through disengagedSmc1/3 hinge domains can be used to entrap DNA within cohesin rings, Sara Cuylen for helpwith figures, and all our colleagues for suggestions and discussions.



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Cohesin: Its Roles and Mechanisms - Haering Lab · 2014-08-12 · ANRV394-GE43-22 ARI 10 October 2009 10:50 INTRODUCTION The segregation of sister chromatids to oppo-site poles of