Article

ISSN 0006-2979, Biochemistry (Moscow), 2024, Vol. 89, No. 4, pp. 601-625 © Pleiades Publishing, Ltd., 2024.

601

REVIEW

Cohesin-Dependent Loop Extrusion:

Molecular Mechanics and Role in Cell Physiology

Arkadiy K. Golov

1,2,a

* and Alexey A. Gavrilov

1,b

Institute of Gene Biology, Russian Academy of Sciences, 119334 Moscow, Russia

Technion – Israel Institute of Technology, 3525433 Haifa, Israel

e-mail: golovstein@gmail.com

e-mail: aleksey.a.gavrilov@gmail.com

Received October 21, 2023

Revised December 29, 2023

Accepted February 15, 2024

Abstract— The most prominent representatives of multisubunit SMC complexes, cohesin and condensin, are best

known as structural components of mitotic chromosomes. It turned out that these complexes, as well as their bac-

terial homologues, are molecular motors, the ATP-dependent movement of these complexes along DNA threads

leads to the formation of DNA loops. In recent years, we have witnessed an avalanche-like accumulation of data on

the process of SMC dependent DNA looping, also known as loop extrusion. This review briefly summarizes the cur-

rent understanding of the place and role of cohesin-dependent extrusion in cell physiology and presents a number

of models describing the potential molecular mechanism of extrusion in a most compelling way. We conclude the

review with a discussion of how the capacity of cohesin to extrude DNA loops may be mechanistically linked to its

involvement in sister chromatid cohesion.

DOI: 10.1134/S0006297924040023

Keywords: cohesin, SMC complexes, loop extrusion, cohesion, DNA gripping state

Abbreviations: CAR, cohesin associated region; E-P, en-

hancer-promoter (interactions); FRET, Förster resonance en-

ergy transfer; HAWK,HEAT protein associated with Kleisin;

SMC,structural maintenance of chromosomes.

* To whom correspondence should be addressed.

INTRODUCTION

Cohesin is a protein complex, which is absolute-

ly essential for reproduction of eukaryotic cells [1, 2].

First cohesin subunits were discovered more than

25years ago as factors participating in pairing of sis-

ter chromatids in mitosis [3-5]. It has been found out

later that this phenomenon termed ‘cohesion’ is based

on the fact that the pairs of sister chromosomes af-

ter replication end up being threaded through multi-

ple cohesion complexes (each of which having ring-

shaped structure with a relatively large intersubunit

pore), similar to two threads passing through a se-

ries of beads (Fig. 1a) [6-8]. Maintenance of cohesion

during the G2-phase of cell cycle and its controlled re-

lease in anaphase occurring due to proteolysis of the

RAD21 subunit of cohesin ensures correct attachment

of spindle microtubules to kinetochores and subse-

quent equal distribution of genetic material between

the two daughter cells [9].

No less important activity of cohesin besides co-

hesion is its ability to form DNA loops via the mecha-

nism called extrusion [2, 10, 11]. Extrusion begins with

cohesin binding to small DNA fragment followed by

the ATP-dependent movement of the complex along

the DNA resulting in processive pulling of the flank-

ing DNA inside the growing loop, the length of which

could eventually reach hundreds of thousands of base

pairs (kbp). Theoretically flanking DNA can be contin-

uously pulled inside from one side of the loop held by

the complex (in the case of unidirectional extrusion) or

from both sides (in the case of bidirectional extrusion).

Extrusion is typical not only for cohesin, but for an en-

tire group of protein complexes, known as SMC com-

plexes (structural maintenance of chromosomes pro-

teins), with cohesin being one of the representatives

of this group [12-14]. Synergistic activity of the typeII

DNA topoisomerases and SMC-dependent extrusion is

required for post-replicative individualization of sis-

ter genomes in all cells (prokaryotic and eukaryotic).

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Fig. 1. Cohesin structure and its participation in sister chromatid cohesion. a)Topological loading of cohesin rings onto sis-

ter chromatids during cohesion. b)Schematic representation of core trimer forming the cohesin ring; recruitment of auxiliary

HAWK subunits to the core trimer. c)Dimerization of head domains of SMC subunits required for ATP hydrolysis.

Extrusion can also facilitate orderly compaction of DNA;

formation of mitotic chromatids in prophase/metaphase

in the vertebrate cells is the best example of such com-

paction. Chromatids are elongated structures consist-

ing of the densely packed chromatin loops anchored

on the proteinaceous axial core. The main structural

component of this axis is condensin– SMC complex re-

sponsible for mitotic loop extrusion, which eventually

results in formation of chromatids. The cohesin-depen-

dent loop extrusion contributes to individualization of

eukaryotic chromosomes, in many cases is responsible

for chromatin compaction and also has a number of

secondary functions.

Cohesion, unlike extrusion, is typical exclusively

for cohesin, and not for any other representatives of

SMC complexes[1,2]. Cohesive activity likely emerged

in the primordial cohesin during early eukaryogen-

esis [15], this acquisition did not result, however, in

the loss of extrusive activity of the complex [16-18].

This commonly accepted scenario raises a number of

interesting questions on mechanistic, functional, and

evolutionary interrelationships between the cohesion

phenomenon and the process of loop extrusion.

In this review we summarize current under-

standing of cohesin-dependent loop extrusion, its role

in cellular processes, and its molecular mechanisms.

We also briefly discuss disparate data indicating pos-

sible mechanistic relationship between extrusion and

cohesion. Detailed description of the cohesin complex

structure and principles of its interactions with chro-

matin can be found in the first part of the review pub-

lished in the same issue of the journal [19].

STRUCTURE OF COHESIN COMPLEX

Cohesin is a protein complex with a ring-shaped

structure, which is based on the trimer of core pro-

teins: SMC1 (Smc1)

, SMC3 (Smc3), and RAD21 (Scc1).

All three proteins have elongated shape and interact

with each other through terminal globular domains

(Fig. 1b). Such organization results in formation of

the extensive intersubunit pore, which is able to let

through globular particles with diameter up to around

10 nm [20, 21]. Presence of the intersubunit pore makes

possible topological entrapment of DNA within the

complex with DNA being threaded through the protein

ring [6-8].

SMC1 and SMC3 subunits are paralogs belong-

ing to the family of ATPases called SMC proteins [22].

SMC1 and SMC3 form a stable V-shaped heterodimer

via homotypic interaction between the hinge domains

of two subunits [20,23]. Head domains, which are lo-

cated at the opposite end of the rod-shaped molecule

from the hinge domains, are responsible for ATPase

activity. In the presence of ATP intermittent engage-

ment of the head domains of two SMC subunits occurs,

such dimerization is required for hydrolysis of bound

In the paper names of human proteins are presented in the main text; names of Saccharomyces cerevisiae homologs are

shown in parenthesis (at first mention).

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ATP molecules (Fig. 1c). The hinge and head domains

of each of the SMC subunits are separated by the long

and relatively flexible coiled-coil arm domain. Flexibil-

ity of the arm domain is to a large degree associated

with the presence of evolutionary conserved defect in

the regular coiled-coil structure, the so-called elbow

region (Fig. 1b). Bending of ‘elbows’ has rather large

amplitude; and simultaneous bending of elbow sites

in both SMC subunits could facilitate direct physical

interaction of the hinge and head domains of the com-

plex. The RAD21 protein, also called kleisin subunit,

forms a constant bridge between the head domains of

the two SMC subunits, thus closing the ring structure.

Auxiliary subunits belonging to the family of

HAWK proteins (HEAT protein associated with Klei-

sin) including STAG1/2

 (Scc3), NIPBL (Scc2), and

PDS5A/B (Pds5) bind to the core trimer [16, 20, 23].

Theprimary site of interaction of the HAWK subunits

with the core trimer is the kleisin subunit, however,

only STAG1/2 forms a stable contact with RAD21 and,

hence, is a constitutive component of the complex.

NIPBL and PDS5A/B compete for the shared binding

site at the kleisin subunit, and both these proteins

interact transiently with the stable cohesin tetramer

(SMC3–SMC1–RAD21–STAG1/2) [24-26]. Therefore, at

each particular moment the complex could contain

one (STAG1/2) or two (STAG1/2 + NIPBL or STAG1/2 +

+ PDS5A/B) HAWK subunits(Fig.1b). Binding of NIPBL

to the core cohesin complex (and presence of DNA)

isrequired for effective hydrolysis of ATP, replacement

of NIPBL with PDS5A/B dramatically changes activity

of the complex [26].

CELLULAR CONTEXT

OF COHESIN-DEPENDENT LOOP EXTRUSION

Cohesin-dependent loop extrusion throughout

the cell cycle. Cohesin-dependent extrusion is realized

in eukaryotic cells throughout interphase and mitosis

(Fig.2a). Various estimates indicate that in the G1-phase

vertebrate cells there are around 100,000 cohesin rings,

and this number doubles in the G2-phase [27, 28].

Nucleoplasm of the cells in G1-phase contains two co-

existing subpopulations of cohesin complexes of ap-

proximately the same size, which are in dynamic equi-

librium: (1) freely diffusing cohesin and (2) cohesin

involved in extrusion (with chromatin residence time of

about 10-30min in vertebrate cells and around 1min

in yeast cells) [28-31]. When cohesion is established

in the S-phase a third subpopulation emerges: stably

bound cohesive rings excluded from extrusion initia-

tion/termination cycle [28, 29, 32]. Complete arrest of

cohesin-dependent extrusion occurs during mitosis. In

vertebrates phosphorylation of the HAWK subunits in

prophase induces termination of cohesion in chromo-

some arms; most likely this phosphorylation also leads

to dissociation of extruding cohesin from DNA [29,33].

Resumption of cohesin-dependent extrusion occurs at

the end of telophase [34,35]. In the budding yeasts the

cohesin-dependent extrusion is realized in mitotic cells

up to the start of anaphase [36], when the separase-de-

pendent proteolysis of the kleisin subunits results in

degradation of the whole cellular pool of cohesin com-

plexes and is resumed only at the end of G1-phase with

restoration of the population of intact cohesin rings

[4,36].

Genomic distribution of initiation sites. Initi-

ation of the cohesin-dependent loop extrusion is not

strictly localized to particular genomic sites, howev-

er, there are some preferences in extrusion complex

binding to DNA. Centromeres and regions adjacent to

them are examples of such sites [8, 26]. Initiation of ex-

trusion, however, also occurs constantly outside of the

centromere regions. It has been assumed up until now

that initiation of the cohesin-dependent extrusion in

the chromosome arms is mainly associated with open

chromatin [37-39]. However, this model is questioned

now. The recently reported experimental data on the

genomic distribution of cohesin binding, as well as the

results of computer modeling indicate that probability

of extrusion initiation is distributed evenly along the

genome outside of the centromeres [40].

WAPL and PDS5A/B are negative regulators of

processivity, NIPBL – positive. The final size of the

loops formed in the process of extrusion is determined

by processivity of the cohesin complexes and genom-

ic distribution of the extrusion pause sites [36, 41-45],

itvaries from several dozens of kb in yeasts [44, 46] to

hundreds of kb in vertebrates[41, 42].

Processivity of the cohesin-dependent extrusion is

suppressed by the HAWK subunit PDS5A/B, as well as

by the WAPL protein recruited by this subunit. Deple-

tion of PDS5A/B and WAPL (together or individually)

significantly increases chromatin residence time of

the extruding cohesin, as well as length of the form-

ing DNA loops [36, 41, 42, 44,45]. Interestingly enough,

both these proteins also participate in non-proteolyt-

ic termination of cohesion as a part of the so-called

prophase cascade: recruitment of the WAPL protein

to cohesin complexes containing PDS5A/B subunit re-

sults in non-proteolytic opening of the protein rings

in the prophase cells of vertebrates and their removal

from the chromosomal arms [47,48]. This dual activity

Vertebrate genomes generally encode a pair of somatically expressed paralogs for both Scc3 and Pds5 HAWK proteins:

STAG1/STAG2 and PDS5A/PDS5B. These paralogs in the majority of cases are structurally and functionally equivalent, hence,

here and further in the text the designations STAG1/2 and PDS5A/B are used.

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Fig. 2. Cohesin activity throughout the cell cycle and chromatin compaction due to SMC-dependent loop extrusion. a)Intact

cohesin complex quantity and their activity throughout mitotic cycle in vertebrate(1) and S. cerevisiae(2) cells. b)Metaphase

chromosomes of vertebrates are formed due to extrusion activity of condensin complexes eventually accumulated in axial struc-

tures. Typical X-shaped structure is maintained due to residual cohesion in centromeres of two metacentric sister chromosomes.

c)Compact chromatid-like “vermicelli” structures formed due to cohesin-dependent loop extrusion in interphase vertebrate

cells with suppressed WAPL activity. Cohesin is the primary structural component of the axial structures in such chromosomes.

ofPDS5A/B and WAPL implies existence of mechanis-

tic relatedness between the process of loop extrusion

and the phenomenon of cohesion.

Another HAWK subunit, NIPBL, on the contrary,

is a positive regulator of the extrusion processivity.

NIPBL is commonly considered as a cohesin loader;

however, this point of view probably requires recon-

sideration as the new data have been reported demon-

strating that NIPBL could be dispensable for the pri-

mary loading of cohesin onto chromatin [49]. It has

been firmly established that recruitment of NIPBL to

the complex is necessary for active extrusion: func-

tional depletion of NIPBL results in suppression of co-

hesin-dependent loop interactions in vertebrates and

inhibition of the cohesin translocation from the pri-

mary loading sites in yeasts [26, 42, 50]. During each

round of extrusion initiation/termination cycle NIPBL

is repeatedly recruited to and released from the com-

plex: chromatin residence time of NIPBL subunit in

the vertebrate G1-cells is around 1 min, which is an

order of magnitude less than average duration of each

round of extrusion [25, 31]. During NIPBL absence its

binding site can be occupied by the PDS5A/B subunit

recruiting WAPL, which, likely, leads to extrusion ter-

mination by not yet elucidated mechanism [2].

Cohesin-dependent interphase extrusion, unlike

mitotic condensin-dependent extrusion, usually does

not result in formation of condensed chromatid-like

structures with proteinaceous axial cores to which

DNA loops are anchored. This can be a consequence

of low processivity of the cohesin-dependent extru-

sion. Suppression of activity of PDS5A/B and WAPL in

the vertebrate cells results in interphase condensation

of chromatin accompanied by the formation of micro-

scopically visible elongated structures with axial cores

containing cohesin [41, 42, 51]. Such compact structures

with characteristic shape resembling metaphase chro-

matids (Fig.2b) have been called ‘vermicelli’(Fig.2c).

Interphase ‘vermicelli’ in a structural sense is simi-

lar to chromatids formed in the meiosisI prophase[52].

Compaction of meiotic chromosomes is achieved by the

extruding activity of cohesin that eventually accumu-

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lates in the axial structures, which later become an im-

portant component of synaptonemal complex. Discov-

ery of cohesin-mediated formation of the condensed

structures similar to metaphase chromatids in the

meiosis I prophase became one of the early indica-

tions that extrusion of DNA loops might be a universal

activity of all SMC complexes [51,53].

Site-specific arrest of loop extrusion. Cohesin-

dependent extrusion differs from extrusion mediated

by other SMC complexes by the ability for a regulat-

ed arrest at the specific genome loci. It is not known

exactly whether such arrest is a temporary pause or

final termination of extrusion. In any case, stability of

the formed DNA loop is maintained by the extrusion

complex for a certain time after arrest until dissoci-

ation of the complex from DNA [31, 54]. At least two

mechanisms of site-specific arrest of extrusion cohes-

in complexes have been described: CAR-dependent

(Cohesin Associated Region) and CTCF-dependent.

Thefirst one, which is evolutionary more ancient and

typical for the cells of lower eukaryotes, is associated

with extended genome regions of cohesin accumula-

tion, CAR-regions [44, 46, 55]. CAR-regions are prefer-

ably immunoprecipitated by the antibodies against

cohesin subunits and, as a rule, are located at the

3′-ends of the convergently transcribed genes [56, 57].

The second mechanism of extrusion arrest associated

with activity of the insulator protein CTCF is realized

in the vertebrate cells [58, 59]. Blocking of extrusion

complexes at the CTCF-sites results in colocalization of

the vast majority of strong cohesin binding sites with

the CTCF binding sites in the vertebrate cells.

CAR-dependent and CTCF-dependent mechanisms

of extrusion arrest have much in common both

with each other and with the processes of stabiliza-

tion of cohesive ring binding to chromatin (Fig. 3a).

In both cases arrest is realized due to the fact that

the blocking sites favor replacement of NIPBL with

PDS5A/B[44, 58]. This replacement is facilitated by the

ESCO1(Eco1)-dependent acetylation of the SMC3 pro-

tein occurring at the arrest sites [43, 45]. Acetylation

of the SMC3 subunit at the conserved lysine residues

K105/K106 (K112 andK113– in yeasts) suppresses ex-

trusion activity of cohesin likely due to decrease in

affinity of the complex to NIPBL[60,61]. Additionally,

acetylation inhibits activity of the WAPL protein [62],

which, as has been mentioned above, removes from

chromatin not only the topologically engaged cohes-

in complexes, but also complexes participating in ex-

trusion [41, 42, 51]. Interestingly enough, stabilization

of the cohesive rings on chromatin in the G2-phase

occurs due to acetylation of SMC3 at the same amino

acid residues (in many species an auxiliary protein so-

rorin also participates in stabilization). Acetylation fa-

cilitating stabilization of cohesion is established in the

S-phase co-replicatively due to activity of the ESCO1

paralog, acetyltransferase ESCO2 (Eco1), and it also

suppresses activity of WAPL towards the acetylated

complexes.

Existence of the genomic elements blocking extru-

sion results in accumulation of cohesin complexes in-

teracting with CAR-elements and CTCF-sites, as well as

in formation of interphase chromatin typical folding

patterns: structural chromatic loops anchored in the

mentioned genomic elements and topologically-associ-

ated domains (TADs) (Fig.3,b andc) [44, 46, 58, 59,63].

In vertebrates the N-terminal fragment of CTCF is re-

sponsible for the arrest of extruding cohesin complex-

es, as well as for inhibition of the activity of WAPL sub-

unit [58, 59,64]. Steric characteristics of CTCF molecule

bound to the binding site allow effective interaction

of its N-terminal fragment with cohesin only if the

extrusion complex approaches the CTCF-motif from

the side of the 3′-end (N-terminal zinc-fingers of CTCF

bind there to DNA); this interaction blocks extrusion

and stabilizes cohesin at the CTCF-bound site (Fig.3a).

At the same time when extrusion complex approach-

es CTCF-motif from the side of the 5′-end it does not

cause prolonged stalling of cohesin. These features of

protein–protein interactions result in the peculiar reg-

ularity in orientation of the CTCF-motifs located at the

bases of structural loops and at the boundaries of to-

pological domains in vertebrates: pairs of CTCF-motifs

located at the bases of structural loops are general-

ly oriented in such a way that their 3′-ends face each

other (convergent orientation); at the same time sev-

eral CTCF-motifs (at least a pair) that are typically

present within each TAD boundary face interior of the

closest topological domain with their 3′-ends (divergent

orientation) (Fig.3,b andc)[63,65-67].

Interplay with transcriptional apparatus of

the cell. Why CAR-sites that stop extrusion in the

yeast cells are located at 3′-ends of the convergently

transcribed genes? Theoretically this could be a con-

sequence of the fact that collision of the transcribing

polymerase with extruding cohesin complexes moving

in the opposite direction results in translocation of

the latter to the end of the transcriptional units. This

model is in agreement with the fact that turning off

transcription leads to the removal of cohesin from the

CAR-sites[56].

The mechanisms underlying transcription-depen-

dent accumulation of cohesin rings at the 3′-end of the

genes are operating not only in yeast cells, they are

universal. For example, despite the fact that the verte-

brate cells do not have classic CAR-sites, simultaneous

depletion of the CTCF and WAPL results in relocation

of cohesin complexes from the CTCF-sites to the 3′-ends

of actively transcribing genes [38, 40]. These new co-

hesin-bound sites differ significantly from the original

genomic peaks colocalized with CTCF; they comprise

extended regions several kbp in length appropriately

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Fig. 3. Mechanisms of cohesin-dependent extrusion arrest and chromatin folding patterns emerging as a result of extrusion

block and subsequent stabilization of extruding complexes. a)CAR-dependent and CTCF-dependent arrest of loop extrusion.

Collision of cohesin with an individual site likely converts bi-directional cohesin-dependent extrusion into unidirectional.

ESCO1(Eco1)-dependent acetylation of SMC3 subunit (acetylation of SMC3 is shown as a yellow dot) plays an important role

inarrest of extrusion and in protection of the stalled complex from WAPL. b)Structural loops with CAR- or CTCF-sites locat-

ed attheir bases are formed due to complete (two-sided) arrest of extrusion. c)Distribution of CAR- and CTCF-sites along the

genome predetermines formation of typical supranucleosomal chromatin folding patterns: structural loops and topologically

associated domains.

named ‘cohesin islands’. The islands, similarly to the

yeast CAR-sites are predominantly located at the 3′-ends

of convergently transcribed genes; their formation can

also be prevented by inhibition of transcription [38].

Emergence of cohesin islands is coupled with forma-

tion of cohesin-dependent chromatin loops between

the pairs of neighbouring islands, which makes their

homology with CAR-sites even more apparent [40].

A number of observations contradict the simplis-

tic notions according to which RNA polymerase di-

rectly interacts with extrusion complexes. One of the

alternative hypotheses suggests that RNA-polymerase

relocates the topologically loaded cohesin rings not

participating in extrusion to the 3′-ends of the genes,

and those, in turn, block the extrusion process [44, 68].

The following facts speak in favor of such mechanism:

(i) preferable localization of CAR-sites at the 3′-ends

of only convergently oriented genes (not at 3′-ends of

each and every active gene), (ii) kinetics of accumu-

lation of cohesin complexes at the CAR-sites during

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cell cycle and temporarily delayed kinetics of loop

formation between the CAR-sites [44], and (iii) abil-

ity of extruding SMC complexes to bypass large chro-

matin-bound protein obstacles, including transcribing

RNA polymerase, demonstrated in vitro[69].

It is likely that the transcribing RNA polymerase

on its own does not represent a significant barrier

for the extruding cohesin complexes, however, active

promoters, which, in addition to RNA polymerase, re-

cruit a large number of auxiliary proteins involved in

initiation, may constitute such barriers. The known

phenomenon of the promoter-associated topological

insulation supports the notion that promoters hinder

movement of extruding cohesin complexes approach-

ing from both directions [40, 70, 71]. Promoters are

less effective extrusion barriers than CAR-regions and

CTCF-sites. Moreover, the cohesin complexes stopped

at promoters are not stabilized via the ESCO1/2-depen-

dent acetylation; that is why promoters are general-

ly not localized at the bases of metastable DNA loops

held by cohesin.

ROLE OF COHESIN-DEPENDENT

LOOP EXTRUSION IN CELL PHYSIOLOGY

Post-replicative individualization of sister

chromosomes and condensation of mitotic chromo-

somes. Individualization of sister chromosomes is a

primordial function of SMC complexes [1,2]. Mechan-

ics of the process of replication of double-helical DNA

molecule in the cells inevitably leads to the certain

degree of topological linkage between two newly syn-

thesized sister DNA threads [72, 73]. Post-replicative

individualization relies on SMC-dependent extrusion,

which directs activity of the typeII topoisomerases to-

wards decatenation of the topologically linked newly

replicated genomic DNA molecules and facilitates spa-

tial separation of the unlinked DNA threads [1, 2, 74].

In the vast majority of cases, bacterial and archaeal

genomes encode a single SMC complex (belonging to

one of the two classes: Smc–ScpAB or MukBEF), main

function of which is exactly post-replicative individ-

ualization of sister chromosomes [1, 2, 75]. In eukary-

otic cells this process is realized through the comple-

mentary activities of two SMC complexes: cohesin and

condensin. In addition to individualization, these com-

plexes also participate in compaction (condensation)

of mitotic chromosomes, which enables relocation of

chromosomes to the spindle poles in anaphase.

Extrusion activity of condensin is suppressed in in-

terphase [76-79], at the same time, throughout G2-phase

cohesin not only maintains cohesion, but also partici-

pates in extrusion, which ensures individualization of

sister chromosomes (to a large extent) even before the

beginning of mitosis[17, 80,81]. At the moment when

the bulk of cohesin rings (in prophase– in vertebrates

and many other eukaryotes, and in anaphase in S. cer-

evisiae) is removed from the chromatin, sister chromo-

somes are already separated from each other to a large

extent and topological links are likely preserved pre-

dominantly at the sites of cohesion: centromeres, ribo-

somal repeats, CAR-regions, and CTCF-sites[17, 80,81].

Removal of the residual topological links and ultimate

individualization of sister chromosomes rely on mito-

sis-specific condensin-dependent extrusion [17,82,83].

Extrusion performed by condensin and cohesin

also ensures mitotic (and meiotic) chromatin compac-

tion (Fig.2,bandc); however, contribution of each of

the complexes differs in the cells of different organ-

isms [1,2]. In particular, cohesin contributes substan-

tially to condensation in the budding yeasts, where the

cohesin-dependent extrusion continues up to the start

of anaphase [44, 84, 85]; at the same time, in vertebrate

cells in which cohesin binding in chromosomal arms is

already terminated by the end of prophase, formation

of compact mitotic chromosomes depends primarily

on condensin [76, 86]. Highly processive activity of

condensin in mitotic cells of vertebrates results in for-

mation of chromatids– compact elongated bodies with

chromatin loops anchored on the axial proteinaceous

core with condensins (vertebrate genomes encode two

types of condensin complexes) and topoisomerase II

being the main constituent of it [87].

It is noteworthy that the processive extrusion ac-

tivity of cohesin can result in formation of chroma-

tid-like structures outside of mitosis. In particular, as

has been mentioned above, cohesin-dependent for-

mation of the chromatid-like “vermichelli” structures

occurs in the interphase cells with suppressed activi-

ty of the WAPL, and axial core of such structures con-

sists of cohesin rings [41, 42,51] (Fig.2c). In the same

way, meiotic chromosomes including transcriptional-

ly active lampbrush chromosomes comprise elongat-

ed structures with chromatin loops originating from

axial cores that have cohesin meiotic variants and

topoisomerase II in their composition in addition to

other proteins [52].

Maintenance of decatenated state of the genome.

Interphase cohesin-dependent extrusion also ensures

specific non-equilibrium folding of chromosomal DNA.

Firstly, the constantly ongoing rounds of extrusion

increase frequency of local cis-interactions and to a

certain extent suppress long-range cis- and trans-inter-

actions [41, 46, 88]. Suppression of trans-interactions,

maintained to some extent by cohesin-dependent ex-

trusion, manifests itself in the formation of more or

less defined chromosome territories in the interphase

eukaryotic cells [89]. Actively maintained bias towards

local DNA contacts in the interphase genome, as will

be discussed later in the review, facilitates accuracy of

DNA repair processes.

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Fig. 4. Cohesin-dependent loop extrusion in the vicinity of double-strand breaks (according to the data reported by

Arnouldetal.[102]). Emergence of double-strand breaks(1,2) leads to the formation of new sites of cohesin-dependent ex-

trusion arrest at the break site(3), to the spread of the γH2AX-signal from the break site along the topological domain due to

unidirectional extrusion(3-5), and to the repositioning of the break site to the interior part of the chromosome territory(5).

The second global consequence of the interphase

cohesin-dependent extrusion is maintenance of genom-

ic DNA in decatenated state [90-92]. Similar to decat-

enation of sister chromosomes, cohesin directs ac-

tivity of topoisomerase II towards resolving, but not

establishing of the interchromosome links. Decatenat-

ed chromatin state facilitates realization of the DNA

template dependent reactions in interphase [93] and

individualization of non-sister chromosomes in mitotic

prophase [85].

Double-strand break repair. Cohesin activity en-

ables accurate DNA damage repair, primarily repair of

double-strand breaks [94-96]. In addition to cohesion,

which facilitates search of homologous recombination

partner in G2-phase of the cell cycle [94, 97], loop ex-

trusion is also an important component of the repara-

tive activity of cohesin.

Firstly, the interphase cohesin-dependent extru-

sion creates and actively maintains specific non-equi-

librium folding of eukaryotic chromatin characterized

by suppression of trans DNA contacts and long-range

cis DNA contacts [41, 46, 88]. Such bias towards local

DNA interactions enables fast and errorless repair of

double-strand breaks. This bias facilitates search for

partners during non-homologous end joining (NHEJ)

DNA repair[96], and also decreases the probability of

ectopic recombination during the recombinational re-

pair [98,99].

Moreover, cohesin is selectively recruited to the

double-strand breaks and participates in their repair

presumably by limiting diffusion of the break ends and

by blocking their interaction with other chromosomes

[96, 98, 100,101]. A number of recent studies showed

a clear connection between this function of cohesin

and its extrusion activity [99]. It has been suggested

that a double-strand DNA break can block movement

of a cohesin extrusion complex in a manner similar to

CAR-regions and CTCF-sites. Bidirectional extrusion is

converted to unidirectional extrusion upon collision

with a double-strand break site, thus a loop held by

an actively extruding cohesin complex is formed on

each side of the break with a break end being one of

the bases of this loop (Fig. 4). Such configuration ex-

plains how exactly cohesin limits break end diffusion

and recruits them to the inner part of the chromosome

territory [99]. This process can be likened to the for-

mation of axial structures in metaphase chromosomes

via the condensin-dependent loop extrusion. Despite

the fact that interphase extrusion does not lead to the

formation of chromatid-like structures in the wild type

cells, the bases of the cohesin-associated loops still

tend to be located in the inner part of the chromosome

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territory somewhat similar to the bases of condensin-

formed loops of the mitotic chromosomes. It is likely

that extrusion exerts similar effect on the ends of dou-

ble-strand breaks, which become bases of the cohes-

in-dependent loops. Restriction of break end diffusion

and their trans-interactions can facilitate accurate res-

toration of the DNA thread integrity in both NHEJ re-

pair pathway and reparative recombination pathway.

Finally, extrusion is also important for the signal-

ing cascades associated with double-strand breaks, in

particular for the spread of γH2AX mark (phosphor-

ylated variant form of H2AX histone) involved in re-

cruitment of repair factors to the genomic region

surrounding the break site (Fig. 4) [102-104]. Unidi-

rectional extrusion ensures systematic recruitment of

the break ends to the surrounding genomic fragments

residing within the same topological domain, which,

in turn, enables the spread of γH2AX-signal from the

break site. The thing is that activity of the enzymes re-

sponsible for the phosphorylation of the H2AX histone

(primarily of ATM kinase) is localized strictly to the

site of DNA damage itself, and in order for this modi-

fication to spread several kbp away in both directions

physical recruitment of these loci to the break site is

necessary. Such recruitment is enabled by the cohesin-

dependent extrusion.

Regulation of transcription in vertebrates. In-

terphase cohesin-dependent loop extrusion is also im-

portant for fine tuning of transcriptional activity in

vertebrates. Cause-and-effect relationships between

the supranucleosomal DNA folding and transcription-

al activity are established in vertebrates due to the

existence of activating remote regulatory elements,

enhancers, in their genomes

 [105, 106]. The general-

ly accepted model of enhancer activity assumes that

the enhancer-activated initiation of transcription is

coupled with the physical interaction between the en-

hancer and promoter of its target gene. Prevalence of

physical interactions between enhancers and promoter

of their target genes, enhancer–promoter (E–P) loops,

in vertebrate cells have been experimentally shown at

the genome-wide scale in recent years [107-109].

Up until recently the notion of the key role of co-

hesin-dependent extrusion in the formation of E–P

loops prevailed [110, 111]. New experimental data,

however, contradict this notion: depletion of cohesin

and suppression of interphase extrusion do not result

in a breakdown of the majority of E–P loops; forma-

tion of these structures, most likely, does not depend

on extrusion [108, 112, 113]. It cannot be ruled out that

cohesin-dependent extrusion is, nevertheless, import-

ant for activity of at least some specific types of en-

hancers – primarily a small group of enhancers con-

taining CTCF binding sites within them [112, 114, 115].

Despite the fact that suppression of the cohes-

in-dependent extrusion in interphase vertebrate cells

does not perturb E–P loop landscape in general, it

causes reproducible transcriptional changes. Although

the observed effects are mostly marginal, hundreds of

genes change the expression level [88, 108]. The ob-

served changes in transcription can be explained by

disturbance of activity of CTCF-associated enhancers

in only a few cases; at least two alternative mecha-

nisms could explain bulk of the observed changes:

(1)insulation effect of TAD boundaries and (2)cohes-

in-dependent suppression of promiscuous interactions

between the active genomic regions.

The majority of topological domains and their

boundaries in vertebrates represent an epiphenom-

enon of cohesin-dependent loop extrusion. Hence,

suppression of extrusion could increase frequency

of E–P interactions between the regulatory elements,

which under normal conditions are separated by the

insulating TAD boundaries. Increased intensity of such

ectopic E–P contacts likely results in the changes of

transcriptional output for some genes [88, 112]. Impor-

tance of the regulatory insulation provided by the TAD

boundaries is confirmed by the genetic observations:

genomic rearrangements causing the disruption of to-

pological insulation have been shown to be associat-

ed with the developmental disorders and oncogenesis.

For some model systems it has been even demonstrat-

ed that specifically ectopic activation of transcription

mediates pathological changes caused by the distur-

bances of TAD landscape [116-118].

The last potential pathway connecting loop extru-

sion to transcription is related to the fact that cohes-

in-dependent extrusion suppresses long-range interac-

tions between the active genomic regions. The not-fully

understood from biochemical point of view mecha-

nism facilitates clustering of active chromatin within

the nucleus, and such clustering results in the forma-

tion of active nuclear compartment or A-compartment

at the level of cell population [65,119]. Active chromo-

somal regions separated by tens of millions of bp, as

well as active sites from different chromosomes can

interact within the A-compartment. Shutdown of the

cohesin-dependent extrusion leads to the uncontrolled

increase of interactions between the active genomic

regions [42, 50,88]; it has been suggested that this may

result in decreased transcription of some genes and in-

creased transcription of the others potentially due to

establishment of super long-range E–P contacts within

the A-compartment[88].

In addition to enhancers activating transcription of their target genes, there are silencers that suppress transcription in

vertebrate cells; in this review only enhancers will be discussed, as information on silencers is currently scarce, nevertheless,

all the described regularities most likely can be extrapolated to silencers.

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Hence, loop extrusion in vertebrate cells affects

transcription regulation via three separate pathways:

via participation in topological insulation, via suppres-

sion of excessive genomic compartmentalization, and,

to a lesser degree, via direct involvement in the forma-

tion of E–P loops.

CYCLE OF CONFORMATIONAL CHANGES

OF COHESIN COMPLEX

DURING LOOP EXTRUSION

Hypothesis according to which compaction and

individualization of mitotic chromosomes are asso-

ciated with the extrusion of DNA loops has been sug-

gested more than three decades ago [120]. Slightly lat-

er Nasmyth[53] suggested that SMC complexes might

be the key components of the cellular machinery of

extrusion. Over the years this speculative concept of

SMC-dependent extrusion has found support in the

data on interphase chromatin structure [65, 66, 88], as

well as in the results of computer modeling of mitot-

ic and interphase chromosomes [111, 121]. Eventually

the ability of cohesin, condensin, and SMC5/6 complex

to perform extrusion of DNA loops has been recently

demonstrated directly in the reconstituted invitro sys-

tems[12, 13, 122, 123].

The accumulated data enabled determination of

many specific characteristics of SMC complex-depen-

dent extrusion, particularly cohesin-dependent. It was

found out that extrusion can be realized exclusively

by the NIPBL containing cohesin complex [122, 123].

The speed of cohesin-dependent extrusion in vivo and

in vitro is ~1 kb/s (estimates in different publications

vary from 0.4 to 3kb/s)[33, 88, 122-124]. Only one cycle

of ATP binding/hydrolysis occurs each second during

cohesin-dependent extrusion [123, 124]. High speed

and the step size as large as ~1kb (which is equivalent

to tens of nanometers even for the nucleosome-packed

DNA) distinguishes SMC complexes from other class-

es of DNA translocases (polymerases, helicases, etc.),

speeds of which are lower by several orders of mag-

nitude and typical step size is equal to 1 bp. Despite

the impressive speed of cohesin-dependent extrusion,

even relatively weak forces (<1 pN) applied to DNA

thread can slow down or even completely block the

process [122,125].

Observations of extrusion in vitro additionally

showed that monomeric cohesin ring performs bidi-

rectional extrusion [Fig. 5a (1)][33, 123, 125], while the

closest cohesin homolog– condensin– performs unidi-

rectional extrusion [Fig. 5a (2)] [12, 126]. Directionality

is one of the most interesting characteristics of the

extrusion process; it is intimately connected to the

molecular mechanism of the process. Emerging DNA

loop can theoretically grow due to the pulling of the

DNA thread from one side of the extruding SMC com-

plex, in this case extrusion is called unidirectional or

asymmetric. In this situation two poles of the loop can

be distinguished: fixed (stable) base, or anchor, and

translocating (mobile) base. Another possible variant

of extrusion is bidirectional or symmetric extrusion in

which the loop grows due to the pulling of DNA inside

the loop from both sides of the active protein com-

plex. In the case of cohesin-dependent extrusion the

bidirectional variant of the process is realized. From

the molecular point of view apparently bidirectional

extrusion can be the result of asymmetric activity of

SMC complex coupled with frequent switching of the

movement direction between the individual cycles of

ATP binding/hydrolysis [126-128]. In such a process of

“switching” bidirectional extrusion, in each ATP hy-

drolysis cycle the growing loop has a fixed and a trans-

locating base, but in each subsequent cycle the bases

can switch the roles.

The covalently linked SMC rings can realize extru-

sion in vitro and form loops on par with the wild type

complexes [69, 123]. Moreover, the mutant forms of

cohesin incapable of maintaining cohesion and, most

likely, incapable of topological engagement with DNA,

can nevertheless be capable of loop extrusion in cells

[8, 18]. Hence, opening of the SMC ring is probably

not an essential step of the extrusion process: cohesin

(and other SMC complexes) interact with DNA non-to-

pologically during extrusion (not holding any of the

two bases of the growing loop inside the intersubunit

pore and not forming true topological links with DNA)

(Fig.5b).

Capability of the complex to traverse DNA-bound

particles with sizes manifold larger than the lin-

ear sizes of the complex itself during the process of

loop growth was an unexpected observation during

the study of cohesin-dependent extrusion [69]. It was

shown that during active extrusion cohesin can by-

pass DNA-associated particles with diameter of up

to 200 nm. Interestingly such bypassing of the obsta-

cles occurs with a minimal slowdown of the complex

movement along the DNA. Traversing massive obsta-

cles indirectly indicates that the process of extrusion

has a non-topological nature [69, 129]. Non-topological

nature of extrusion has been also indirectly support-

ed by the following observation: binding of cohesin to

DNA at the bases of chromatin loops formed during

extrusion are disrupted in the permeabilized nuclei in-

cubated in buffer with comparatively mildly increased

ionic strength [130].

Despite the fact that many general characteristics

of the cohesin-dependent loop extrusion have been

documented in the recent years (especially substantial

progress was achieved due to the development of invitro

reconstituted systems of extrusion), molecular mech-

anism of extrusion is still not completely elucidated.

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Fig. 5. Molecular details of SMC-dependent extrusion. a)Bidirectional extrusion realized by cohesin monomers(1), and uni-

directional extrusion realized by the condensin complexes (2). ‘Safety belt’, likely, stabilizes binding of the complex to one

ofthebases of the growing loop during condensin-dependent extrusion. b)Hypothetical modes of SMC complex binding to

DNA ofthegrowing loop during extrusion.

Wedo not know how exactly the cycle of ATP binding/

hydrolysis is coupled with conformational changes of

the complex, and how these changes orchestrate the

growth of DNA loops. Due to considerable interest to

this problem and due to insufficient amount of exper-

imental data, several competing models of the process

have been suggested. Three such models, which are in

greatest agreement with the available structural, ge-

netic, and biochemical data, are: pumping/hold-and-

feed model, Brownian ratchet model, and swing-and-

clamp model. Below, brief description of each of these

models is provided. It must be emphasized that despite

the elegance and impressive explanatory power of

each of the discussed model, none of them is in agree-

ment with the whole entirety of the available experi-

mental data.

Gripping state/DNA clamping. Despite the fact

that the models of extrusion process described be-

low differ in numerous significant parameters, one of

the crucial intermediates of the extrusion cycle– the

so-called DNA ‘gripping state’– is common for all the

models. DNA gripping has been described in the very

recent years as a result of investigation of cohesin

and condensin structures with the help of cryogenic

electron microscopy [61, 126, 131]. It was discovered

that in the presence of NIPBL, double-stranded DNA,

and non-hydrolyzable ATP analogs (or alternatively

if cohesin contains SMC subunits with mutations pre-

venting ATP hydrolysis) cohesin forms specific type

of stable complexes with DNA called ‘DNA gripping

state’. In the gripping state DNA electrostatically binds

to the upper surface of the head domains engaged in

the presence of ATP (Fig.6a). NIPBL interacts with the

arm region of the SMC3 subunit and simultaneously

with the dimerized head domains thus forming a pro-

tein bridge pinning down the DNA thread to the head

domains from above. NIPBL in the described structure

also forms a series of electrostatic contacts with DNA.

The data obtained using FRET (Förster Resonance En-

ergy Transfer) indicate that in the presence of hydro-

lysable ATP the gripping state is rapidly formed and

disassembled [124]. It is likely that this cycle of forma-

tion and disassembly is strictly coupled to the ATP hy-

drolysis cycle: ATP hydrolysis leads to the disruption

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Fig. 6. Cohesin-dependent extrusion according to the pumping/hold-and-feed model. a)Pseudo-topological variant of the ex-

trusion pumping/hold-and-feed mechanism (according to the data reported by the Hearing group [126]). b) Non-topological

variant of the extrusion pumping/hold-and-feed mechanism (according to the data reported by Oldenkamp and Rowland[10]).

c)Exchange of the two bases of the growing loop during pseudo-topological extrusion could enable frequent switch of the move-

ment direction and be manifested as an apparent bidirectionality of the process. In all pictograms direction of DNA thread

relative to the figure plane is shown with ●and+ symbols. DNA sites, which are or were at previous stages dynamic bases of

the loop are shown in red, and stable bases– in blue. Dashed line fragment of DNA thread reflects potentially unlimited size

ofthegrowing loops.

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of the head domain dimer, which contains an extended

DNA-binding groove on its surface. This probably also

leads to the dissociation of NIPBL and eventually to

the release of the clamped DNA fragment. Binding of

a new pair of ATP molecules by the head domains re-

sults in reassembly of the gripping state. The described

coupling of ATP hydrolysis cycle and reversible DNA

binding to cohesin in the gripping state clearly indi-

cates that this structure may play an important role

in the translocation of cohesin along the DNA during

extrusion. However, the described static structure of

DNA gripping state does not tell anything about the

structural rearrangements leading to the directional

movement of cohesin and binding of new fragments of

the DNA thread in each next cycle in the gripping state

formation.

Pumping/hold-and-feed model. One of the prom-

inent structural characteristics of DNA gripping is dis-

engagement of the arm domains of the two SMC sub-

units [61, 132]. It is known that after ATP hydrolysis

the interaction between head domains of SMC subunits

can be rearranged resulting in the formation of the so-

called juxtaposed state (or J-state), which leads to the

closing of intersubunit pore and establishment of tight

interactions between the arm domains along their en-

tire length [133, 134]. There are some indications that

the closing of the intersubunit pore during transition

to the J-state occurs processively from top to the bot-

tom similarly to zipper closing[135, 136]. The pumping/

hold- and-feed model suggests that the translocation

of cohesin complex along DNA is mediated through

repeated cycles of arm domain ‘zipping’ coupled

with the cycles of ATP hydrolysis (Fig. 6a) [136, 137].

The most elaborate version of this model recently

presented by the Haering group[126] is based on the

cryogenic electron microscopy data, as well as data re-

garding entrapment of DNA threads within different

subcompartments of the SMC ring obtained with the

help of thiol-specific complex cross-linking.

This model suggests that one of the DNA-bases

of the growing loop is anchored on STAG1/2 (Fig.6a).

DNA of the loop mobile base is held by NIPBL close to

the SMC head domains. Translocation of the complex

along DNA occurs due to the fact that formation of the

gripping state is mechanically coupled with threading

of the DNA micro loop through the intersubunit pore.

ATP hydrolysis leads to coalescence of the captured

micro loop with the main DNA loop held in the vicin-

ity of the head domains of the complex. Coalescence

results in substitution of the original mobile base of

the loop with a new one located downstream. DNA site

electrostatically bound to the inner surface of the di-

merized hinge domains during initial capture of the

micro loop assumes role of such new mobile base. This

site is handed over to the NIPBL subunit in the course

of ‘zipping’ of arm domains. ‘Zipping’ returns the com-

plex to the initial J-state, while the loop gets longer

than the original one due to the absorption of micro

loop in the course of this process.

There are two principal variants of the pumping/

hold-and-feed model: pseudo-topological variant in

which each of the two bases of the growing loop are

threaded through the cohesin pore while the complex

is in the J-state(Fig.6a), and non-topological variant in

which the entire growing loop interacts with the com-

plex in a non-topological manner (Fig.6b).

The pseudo-topological variant of the model is

in agreement with many experimental observations

made in the reconstructed in vitro extrusion systems.

Firstly, the possibility of efficient micro loop capture

depends of the mechanical tension of the DNA mole-

cule. This explains how even small mechanical forces

(~ 1 pN) are capable of preventing micro loop capture

and therefore of blocking extrusion [122, 125]. Sec-

ondly, in in vitro systems cohesin complexes perform

bidirectional extrusion [123, 125]. Pseudotopological

variant of pumping/hold-and-feed model suggests that

dissociation of the NIPBL subunits from the complex

(occurring periodically during extrusion) can result in

formation of a single subcompartment holding both

bases of the growing loop. It is assumed that interac-

tion of the STAG1/2 subunit with the loop anchor also

can be periodically disrupted (Fig. 6c). Restoration of

binding of the loop bases to the HAWK subunits could

result in the exchange of the two DNA threads, which

is equivalent to the change in extrusion direction.

Hence, the apparent symmetry of cohesin-dependent

extrusion can be explained as a result of periodically

occurring change in the direction of asymmetric pro-

cess. The model also explains why the wild type con-

densin is a strictly unidirectional extruder [12, 126].

Presumably unidirectionality of condensin is ensured

by the additional strength of the loop anchor binding

to the complex; this strength is provided by the ‘safety

belt’– structural feature absent in the kleisin subunit

of cohesin (Fig.5a). Mutations destabilizing the ‘safety

belt’ convert condensin into a bidirectional extruder

similar to cohesin [126].

The pseudo-topological variant of the model,

however, contradicts at least one crucial experimen-

tal observation: the ability of the complex to traverse

massive DNA-binding particles in the course of in vitro

extrusion [69]. In order to resolve this contradiction,

a non-topological variant of the model was suggested

[10]. It was found out that the vast majority of empiri-

cal observations, underlying initial pseudo-topological

variant of the model, could be equally well explained

under assumption that the growing DNA loop is not

threaded through the complex (Fig. 6b). This vari-

ant of the model suggests that the mobile base of the

loop itself is bound to the complex as a small pseudo-

topological loop formed due to interaction of the

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NIPBL subunit with the juxtaposed head domains.

Upon ATP hydrolysis this small loop coalesces with

the micro loop captured inside the intersubunit pore,

next it is tightened to its initial size due to the ‘zipping’

of the arm domains. Such process, unlike the mecha-

nism suggested in the pseudo-topological model, is in

agreement with the observed phenomenon of the co-

hesin complex bypassing massive obstacles during

extrusion.

Paradoxically, non-topological variant of the

pumping/hold-and-feed model explaining the ability

of cohesin to traverse obstacles, cannot explain bidi-

rectional nature of the cohesin-dependent extrusion:

hypothetical exchange of the DNA threads is only pos-

sible inside a single pseudo-topological compartment

holding both bases of the growing loop. The majority of

the attempts to describe molecular details of extrusion

face this fundamental problem: impossibility to recon-

cile in one model the bidirectional nature of extrusion

and the ability of the complex to bypass massive barri-

ers. Pseudo-topological models usually cannot explain

traversing of massive barriers, while the non-topolog-

ical ones cannot be reconciled with the bidirectional

nature of the process. Some authors, however, suggest

that some variants of non-topological models may

under certain assumptions include exchange of DNA

threads, switch of the extrusion direction, and, eventu-

ally, bidirectional nature of the process [128].

Brownian ratchet model. Two other models de-

scribed below belong to the class of extrusion models

based on the bending movement of ‘elbows’ (scrunch-

ing models). These models assume that the ‘elbow’

bending and subsequent engagement of hinge domains

with head domains is somehow coupled with the trans-

fer of the mobile base of the loop from one pair of the

domains to another [134, 138]. Unlike the pumping/

hold-and-feed model in which the key conformational

change coupled with the ATP binding/hydrolysis cycle

is ‘zipping’ of the intersubunit pore, scrunching mod-

els of extrusion assume that the key conformational

change enabling complex translocation is reversible

bending of the ‘elbows’.

This class of models rely on the ability of cohesin

and other SMC complexes to assume conformations in

which the ‘elbows’ are fully bent, and head and hinge

domains are close to each other [134, 139]. Thecryo-

genic electron microscopy data showed that such

bending could be theoretically coupled with ATP bind-

ing and formation of DNA gripping state [60, 61, 131].

Additional structural data obtained with the help of

atomic force microscopy, FRET, covalent cross-linking

of the complexes, and observations of the ability of

mutant complexes to form loops in vitro enabled the

development of two most detailed scrunching extru-

sion models: Brownian ratchet model [127, 131] and

swing-and-clamp model [124].

In the Brownian ratchet model “elbow” bend-

ing results in pulling of pseudo-topological DNA loop

through the cohesin ring. The model was developed

by the Uhlmann group[127, 131] as a result of analy-

sis of one of the first cohesin gripping state struc-

tures. The authors noticed that in the gripping state

structures resolved with the help of cryogenic elec-

tron microscopy STAG1/2 binds to DNA in vicinity to

the clamped site, and that due to the elbow bending,

dimer of the hinge domains is close to the STAG1/2

(Fig. 7a). Additional FRET-experiments demonstrated

(these results were not however confirmed by the later

studies) that proximity between the hinge domains and

STAG1/2 in the complex has constitutive nature. Hence,

Brownian ratchet model suggests that cohesin has two

DNA-binding modules: head module associated with

NIPBL, and hinge module associated with STAG1/2. Un-

like the pumping/hold-and-feed model and the swing-

and-clamp model, the Brownian ratchet model suggest

that both HAWK subunits bind to the mobile base of

the growing DNA loop, while retention of the loop an-

chor site within the complex occurs passively due to

pseudo-topological interaction of the complex with the

DNA loop.

The model assumes that disruption of DNA grip-

ping caused by ATP hydrolysis results in straighten-

ing of the ‘elbows’ and destabilization of interaction

of the STAG1/2-hinge module with DNA. This desta-

bilization eventually leads to the loss of interaction,

however, before the complete disruption of the bond

between the STAG1/2-hinge module and DNA occurs,

the ‘elbows’ have time to straighten up to at least some

extent (Fig. 7a). Elbow straightening results in the

growth of the captured pseudo-topological loop due to

the movement of the complex along one of the DNA

threads– unidirectional extrusion. Pseudo-topological

interaction of the complex with DNA allows complex

to hold the growing loop even after the complete loss

of electrostatic interactions. The growing DNA loop is

held pseudo-topologically inside the intersubunit pore

up to the moment of ATP binding and assembly of the

new DNA gripping state.

It is generally accepted that the elbow straight-

ening is a reversible equilibrium process realized

through the thermal motion; directional nature of ex-

trusion is ensured in this model by the ratchet mech-

anism: power stroke movement always starts at the

gripping state with elbows completely bend. These two

features of cohesin movement in the Brownian ratchet

mechanism are reflected in the name of this model.

Similar to the pseudo-topological variant of the

pumping/hold-and-feed model, the Brownian ratchet

mechanism explains bidirectional nature of the co-

hesin-dependent extrusion by exchange of the DNA

threads of the growing loop, which potentially could

occur in each cycle of ATP binding/hydrolysis at the

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Fig. 7. Cohesin-dependent extrusion according to the ‘scrunching’ models. a) Extrusion according to the Brownian ratchet

mechanism (according to the data reported by Higashietal. [127]). b)Extrusion according to the swing-and-clamp mechanism

(according to the data reported by Baueretal.[124]). Fragments of RAD21 subunit depicted by dashed lines correspond to the

regions in which path of the protein chain is shown arbitrarily to increase figure clarity (in reality HAWK subunits remain

bound to RAD21 during all the presented stages). All other designations as in Fig.6.

stage of passive trapping of the pseudo-topological

loop inside the cohesin ring. Equilibrium character of

the ‘elbow’ extension explains why even weak exter-

nal forces completely block the SMC dependent extru-

sion invitro.

There are however two significant drawbacks of

the Brownian ratchet model: (1) impossibility to recon-

cile pseudo-topological extrusion with experimentally

observed ability of cohesin to bypass massive barriers

[69] and (2) absence of an independent validation for

GOLOV, GAVRILOV616

BIOCHEMISTRY (Moscow) Vol. 89 No. 4 2024

the existence of the stable STAG1/2-hinge DNA-binding

module.

Swing-and-clamp model. Another extrusion mod-

el based on ‘elbow’ bending proposed by the Peters

group [123] suggests that binding of DNA by the hinge

domains and bending of ‘elbows’ (‘swing’) precedes

formation of gripping configuration, hence, this mod-

el was named ‘swing-and-clamp’ [124]. The model is

based on two key observations obtained using FRET

technique: 1) NIPBL protein constitutively interacting

with RAD21 can also form temporary ATP-dependent

contacts with other subunits of the complex and 2) de-

spite the relative freedom of bending/straightening

movements of arm domains, physical interaction be-

tween the hinge and head domains appears to be not

compatible with the gripping; this means that ‘elbows’

during gripping are always in a more or less straight-

ened state. This model suggests non-topological mode

of extrusion with the stable base of the loop anchored

at STAG1/2 (as in the pumping/hold-and-feed mod-

el). Move of the mobile base of the loop relative the

SMCcomplex occurs due to transfer of DNA from the

hinge domains to the head domains during formation

of the DNA gripping state and due to the subsequent

straightening of the ‘elbows’ (Fig.7b). The DNA trans-

fer occurs during the formation of DNA gripping state

and is coupled with the switch in NIPBL–SMC inter-

action pattern. Prior to the formation of the gripping

state NIPBL is bound to the hinge domains, and togeth-

er they hold DNA of the loop mobile base. Assembly

of the gripping state is associated with the transfer of

both the loop mobile base and the NIPBL subunit hold-

ing it from the hinge domains to the head domains;

in the process, hinge domains lose their interaction

with NIPBL. Immediately afterwards straightening of

the ‘elbows’ occurs due to allosteric effects of DNA

gripping formation. It is assumed that prior to ATP hy-

drolysis and subsequent disassembly of the gripping

state the hinge domains bind a downstream DNA site,

which eventually becomes the new mobile base of the

growing loop. Disassembly of the gripping state results

in dissociation of NIPBL from the head domains and

its reverse jump to the hinge domains thus beginning

a new cycle of conformational changes. Hence, con-

trary to the Brownian ratchet model, in the swing and

clamp model the mobile base of the loop is electrostat-

ically bound to one or another DNA-binding surface of

cohesin over the entire duration of the cycle. Such con-

tinuous contact is indispensable for non-topological

maintenance of the growing DNA loop.

In addition to being in agreement with numerous

structural data swing-and-clamp model also provides

clear and specific explanation of how the processes of

elbows bending/straightening are coordinated with the

cycle of ATP binding/hydrolysis and with the transfer

of the DNA thread from the hinge domains to the head

domains. Bending of arm domains in this model is a

prerequisite for the formation of DNA gripping state

and ATP hydrolysis; DNA gripping state formation, in

turn, is coupled with the transfer of the mobile loop

base from one binding site to another. At least partial

expansion of the elbows, which always precedes bind-

ing of the new mobile base by the hinge domains, en-

sures directional movement of the extrusion complex.

It should be mentioned that in the swing-and-clamp

model, like in the Brownian ratchet model, bending/

straightening of the arm domains mediating move-

ment of the cohesin complex along the DNA is an equi-

librium process, which accounts for high sensitivity

of the extrusion speed to external forces [122, 125].

Non-topological nature of extrusion postulated in this

model is in agreement with the ability of extrusion

complex to bypass massive DNA-bound particles [69].

At the same time, the swing-and-clamp model predicts

exclusively unidirectional mode of extrusion, which

contradicts the observations made in the reconstituted

invitro systems [123,125].

COHESION AND COHESIN-DEPENDENT

LOOP EXTRUSION MIGHT BE

MECHANISTICALLY RELATED

From the time of its discovery and up until now

cohesin is better known as a complex ensuring cohe-

sion of sister chromatids through its ability for topo-

logical loading onto DNA. The fact that cohesin is also

a DNA translocase capable of formation DNA loops via

the extrusion mechanism is often considered as a mi-

nor detail, secondary addition to its primary cohesive

function. Ever increasing number of cellular process-

es shown to be reliant on the cohesin-dependent loop

extrusion unambiguously demonstrates the fallacy of

such view: extrusion is along with cohesion one of the

fundamental activities of cohesin. This point of view

is in good agreement with the evolutionary primitive-

ness of extrusion which is known to be a feature of al-

most all SMC complexes [12-14,123]. From phylogenetic

point of view cohesive pairing of sister chromatids has

likely emerged as an adaptation of the primordial co-

hesin to perform an additional function not typical for

other related complexes[15-17]. Thus, one of the unre-

solved mysteries of cohesin biology, namely, whether

cohesion and extrusion are parts of the single molecu-

lar pathway draws even more attention.

Many authors intuitively assume positive answer

to this question [61, 127]. Nevertheless, even the most

general scheme of such a pathway still remains a con-

troversy. Development of the concepts regarding the

nature of this pathway turned out to be closely in-

tertwined with the ideas about the mode of cohesin

binding to DNA during extrusion. The most general

COHESIN-DEPENDENT EXTRUSION 617

BIOCHEMISTRY (Moscow) Vol. 89 No. 4 2024

Fig. 8. Scheme of the hypothetical molecular pathway which includes both cohesin-dependent extrusion and cohesion. a)The

scheme of originally proposed pathway of this kind, suggesting equivalence between topological cohesin loading and extrusion

initiation. b)Scheme of the modified version of such a pathway taking into account the accumulated structural data on extru-

sion process: topological cohesin loading is suggested to be coupled with termination of extrusion. DNA gripping state plays

here a key role in the switch between extrusion and topological engagement.

indication on the existence of mechanistic interplay

between the extrusion and cohesion is the fact that

the PDS5A/B subunit and WAPL protein participate in

both removal of cohesive rings from DNA [47,48], and

in extrusion termination [41, 42, 44, 51]. Initially this

observation prompted a hypothetical scheme in which

extrusion is realized by the cohesin rings topological-

ly loaded onto DNA, and these rings may be converted

into cohesive complexes during replication (Fig.8a).

However, accumulation of new data resulted in re-

jection of this naive model. We are primarily referring

to the structural data indicating that extruding cohesin

complexes are not topologically engaged with DNA [69,

123, 130]. These structural data have later received ge-

netic validation: it was shown that certain mutations

in SMC subunits can suppress ability of the complex

for topological loading not affecting its capacity to ex-

trude DNA loops [8,18]. Today, when it can be assumed

with sufficient certainty that extrusion follows either

non-topological or pseudo-topological mechanism, an-

other concept has come to prominence. According to

this concept cohesin topological loading (and subse-

quent cohesion establishment) is just an alternative

pathway for termination of extrusion-associated ATP

hydrolysis cycle. Many authors suggest that DNA grip-

ping as a key intermediate stage of the ATPase cycle

where the choice between continuation of the ex-

trusion and topological loading eventually resulting

in extrusion termination is made (Fig. 8b) [61, 127].

Therole of the PDS5A/B–WAPL tandem might be then

in tipping the reaction towards termination of extru-

sion cycle and cohesin topological engagement with

DNA [130]. This hypothetical scenario implies that the

ability of the PDS5A/B–WAPL subcomplex to open co-

hesin ring is crucial for two different reactions taking

place in the cell nucleus: cohesin topological loading/

termination of extrusion and removal of topologically

engaged cohesin rings from chromatin.

GOLOV, GAVRILOV618

BIOCHEMISTRY (Moscow) Vol. 89 No. 4 2024

CONCLUSIONS

For many years DNA loop extrusion was more of

a speculative concept; even then though some authors

emphasized that only extrusion could explain a num-

ber of cellular processes such as decatenation of sister

DNA molecules [53]. Last decade was the time of accu-

mulation of first indirect and next direct experimental

confirmations that this process indeed happens in cells,

and SMC complexes plays the central role in this pro-

cess[12, 66, 111,123]. We have arguably witnessed an

extremely rare event: validation of theoretical predic-

tion in biology.

Structural studies of SMC complexes, analysis of

molecular factors affecting SMC-dependent loop extru-

sion, and microscopic observation of extrusion in in vitro

systems significantly clarified our understanding of the

nature of the process. Let us mention the most signif-

icant observations regarding cohesin-dependent loop

extrusion reported in recent years: 1) bidirectional na-

ture of the process [122,123]; 2) non-topological or pseu-

do-topological nature of binding of extrusion complex

to DNA [8, 18, 69, 123, 130]; 3) high speed of loop growth

reaching several kbp per second [33, 88, 122-124]; 4) key

role of NIPBL subunit in the process of active extrusion

[42, 122, 123]; 5) periodic ATP-dependent formation of

DNA gripping state during the cycle of conformational

changes associated with extrusion [60, 61, 131]; 6) partic-

ipation of PDS5A/B and WAPL (recruited by the former)

in termination of the process [41, 42, 44, 51]; 7) arrest of

extrusion at the specific genomic regions, CTCF-sites and

CAR-regions, resulting in formation of metastable loops

anchored in these regions [44, 58, 59]. These results, de-

spite their exceptional importance, are just a series of

more or less disparate facts that do not add up to form

a clear unified picture. The attempts to develop such a

universal picture have been already made [140]. The

most important component of such picture should be

detailed description of molecular rearrangements of the

complex during extrusion. In this review we among oth-

er things described the three most convincing molecular

models of extrusion; each of the presented models being

more or less speculative and in only partial agreement

with the available experimental data. We also address

the intriguing and poorly understood question of mech-

anistic relationships between cohesion and extrusion.

Additional data that would be obtained in the near fu-

ture hopefully can help to fill up the existing gaps in our

understanding of loop extrusion mechanics and enable

creation of a clear, unified, and experimentally ground-

ed model of the process, such model seems to be es-

sential for dispelling the mystery of cohesin, two-faced

Janus of the eukaryotic chromosome biology.

Acknowledgments. The authors are grateful to

S. V. Razin for fruitful discussions of many topics ad-

dressed in the review. The authors also are thankful

toA. V. Golova for help in preparing illustrations.

Contributions. A.K.G. summarizing the data, writ-

ing the first draft of the paper; A.A.G. formulation of

the problem, supervision of the work, editing text of

the paper.

Funding. This work was financially supported by

the Russian Science Foundation (grant no.21-64-00001).

Ethics declarations. This work does not con-

tain any studies involving human and animal sub-

jects. Theauthors of this work declare that they have

noconflicts of interest.

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