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Journal of Cell Science 110, 1345-1350 (1997)
Printed in Great Britain © The Company of Biologists Limited 1997
JCS9577
COMMENTARY
When helicase and topoisomerase meet!
Michel Duguet
Laboratoire d’Enzymologie des Acides Nucléiques, Institut de Génétique et Microbiologie, URA 2225 CNRS, Université Paris-Sud,
91405 Orsay Cedex, France
(e-mail: [email protected])
SUMMARY
Several examples of direct interactions between helicases
and topoisomerases have recently been described. The data
suggest a possible cooperation between these enzymes in
major DNA events such as the progression of a replication
fork, segregation of newly replicated chromosomes, disruption of nucleosomal structure, DNA supercoiling, and finally
recombination, repair, and genomic stability. A first example
is the finding of a strong interaction between T antigen and
topoisomerase I in mammalian cells, that may trigger
unwinding of the parental DNA strands at the replication
forks of Simian Virus 40. A second example is the reverse
gyrase from thermophilic prokaryotes, composed of a
putative helicase domain, and a topoisomerase domain in the
same polypeptide. This enzyme may be required to maintain
genomic stability at high temperature. A third example is the
finding of an interaction between type II topoisomerase and
the helicase Sgs1 in yeast. This interaction possibly allows
the faithful segregation of newly replicated chromosomes in
eukaryotic cells. A fourth example is the interaction between
the same helicase Sgs1 and topoisomerase III in yeast, that
may control recombination level and genetic stability of
repetitive sequences. Recently, in humans, mutations in
genes similar to Sgs1 have been found to be responsible for
Bloom’s and Werner’s syndromes. The cooperation between
helicases and topoisomerases is likely to be extended to many
aspects of DNA mechanisms including chromatin condensation/decondensation.
INTRODUCTION
Several recent examples of interactions of helicases with
topoisomerases suggest a cooperation between these two
classes of enzymes in many aspects of DNA mechanisms (Fig.
1). These include progression of the DNA replication fork, segregation of newly replicated chromosomes, disruption of
nucleosomal structure (especially during transcription), DNA
supercoiling, and finally DNA recombination, repair, and
genome stability.
Early in evolution, the choice was made to have the plectonemic DNA double helix as the carrier of genetic information.
From this initial choice, it was necessary to solve the topological problem of separating the two DNA strands in order
to allow the proper transmission of the genetic information.
Two steps are required for this process: (1) disruption of
hydrogen bonds between the two strands, performed by specialized enzymes called DNA helicases (reviewed by Lohman,
1993); and (2) elimination of all the topological links between
the two strands, performed by DNA topoisomerases (reviewed
by Wang, 1996). These considerations led to the suggestion
that DNA helicases and topoisomerases are both required to
provide the swivel mechanism for DNA replication, postulated
more than three decades ago by Cairns (1963).
Surprisingly, various models for the replisome, a compact
replication complex comprising DNA polymerase/primase and
helicases, did not generally integrate a topoisomerase
(Kornberg, 1978). The first indication of a cooperation between
a helicase and a topoisomerase came from studies on phage
ΦX174 DNA replication. An interaction between the host
encoded rep DNA helicase and the phage gene A protein
product (a sequence specific topoisomerase) allows for strand
separation of the phage DNA replicative form (Scott and
Kornberg, 1978; Duguet et al., 1978).
Key words: Replication fork, Chromosome segregation,
Nucleosome, DNA supercoiling, Recombination, Genetic stability
PROGRESSION OF THE REPLICATION FORK
In the late seventies, it was suggested that the topological
problems arising at a replication fork could in principle be solved
by two different mechanisms (Champoux and Been, 1980;
Forterre et al., 1980). The first consists of the removal of the
positive supercoils arising in front of the replication fork, as the
parental strands rotate. In eukaryotic cells, this may be achieved
indifferently by topoisomerase I or II (Kim and Wang, 1989;
DiNardo et al., 1984), although the former enzyme appears the
more efficient because of its highly processive relaxing activity
(Roca, 1995). In prokaryotic cells, DNA gyrase (topoisomerase
II), efficiently removes positive supercoils, and may function
ahead of the fork (Wu et al., 1988), while topoisomerase I
(protein ω) is probably less involved, as it is unable to bind positively supercoiled DNA (Kirkegaard and Wang, 1985). Fig. 2A
1346 M. Duguet
depicts a hypothetical swivelase (see also Fig. 5) formed by the
association of a helicase and a topoisomerase. In this model, the
two enzymes are part of a large replication complex anchored in
a fixed structure (i.e. the nuclear matrix) and the replicating DNA
is translocated through the protein complex (Kornberg, 1988).
The helicase actively separates the two parental DNA strands
while the topoisomerase, working in front of the helicase, allows
relaxation of positive supercoils in a highly processive manner.
Indeed, a direct interaction between eukaryotic topoisomerase I
and T antigen was recently described, that may trigger DNA
unwinding at the SV40 replication forks (Simmons et al., 1996).
The second mechanism to prevent positive supercoiling at the
replication fork allows newly replicated DNA duplexes to
untangle (Champoux and Been, 1980; Forterre et al., 1980),
instead of rotating the parental duplex. This may be achieved by
a type II topoisomerase working behind the helicase and able to
perform transient double strand breaks in the DNA. Such a
mechanism is likely to occur at the end of replication (see below).
would explain why a defect in a helicase that does not interact
with topoisomerase II does not result in chromosome unstability
(i.e. XPD or ERCC2).
Very recently, two human genes, BLM and WRN (respectively 43% and 38% identical to Rec Q, and 44% and 34% to
SGS-1 over the 400 amino acid sequence of the helicase
region) have been identified as responsible for Bloom’s and
Werner’s syndromes (Ellis et al., 1995; Yu et al., 1996). Similar
to mutations in SGS-1, mutations in BLM and WRN result in
chromosomal breakage, translocations, intra and interchromosomal strand exchange. Furthermore, a reduction in 1 M NaCl
extractible topoisomerase II was found in Bloom’s cells treated
by BrdU (Heartlein et al., 1987).
Finally, it is noteworthy that SGS-1 (and perhaps the human
genes) not only interact in vivo with topoisomerase II, but also
in a distinct pathway with topoisomerase III, an interaction that
is presumably required to reduce the level of recombination
(see section on recombination and genome stability).
SEGREGATION OF NEWLY REPLICATED
CHROMOSOMES
DISRUPTION OF NUCLEOSOMAL STRUCTURE
It has been proposed long ago that proper chromosome segregation presents a major topological problem, both in eukaryotic
and in prokaryotic cells, because of the intertwining of newly
replicated DNA molecules (Sundin and Varshavsky, 1980).
Failure to solve this problem results in breakage of chromosomes, non-disjunction, and eventually cell death (Holm et al.,
1989; Uemura et al., 1987; Spell and Holm, 1994). The essential
role of type II topoisomerases (Topo II in eukaryotes, gyrase or
Topo IV in bacteria) in the untangling of chromosomes has been
well recognized (DiNardo et al., 1984; Adams et al., 1992).
Fig. 2B shows schematically the problem arising when two
forks meet at the end of replication (Champoux and Been, 1980;
Forterre et al., 1980; Wang, 1991; Holm, 1994). As pointed out
by Holm, when the tracking DNA helicases approach each other,
any double helical turn in the parental duplex is converted into
one intertwining between the two daughter molecules. Topoisomerase II removes these intertwinings by its double-strand
breaking/passing/rejoining activity (Wang, 1996). Thus, a
helicase working at a replication fork and a type II topoisomerase
working in association with it, may cooperate to perform chromosome segregation. Until recently, this cooperation remained a
matter of hypothesis. Studies in yeast, however, may have
changed this perspective. Indeed, in a search for proteins interacting in vivo with the C-terminal domain of topoisomerase II in
yeast, Watt and coworkers (1995) used the two-hybrid cloning
strategy (Fields and Song, 1989). They found an interacting
protein named SGS-1, a putative helicase whose mutation was
previously described as a top3 suppressor (Wallis et al., 1989, see
section on recombination). SGS-1 shows strong sequence similarity to Escherichia coli RecQ helicase (Umezu et al., 1990).
Deletion of SGS-1 results in increased chromosomal mis-segregation and high levels of non-disjunction at meiosis I. The authors
also showed that SGS-1 and Topo II likely act in the same chromosome segregation pathway. This latter finding indicates that
chromosome segregation in yeast is probably promoted by a
direct cooperation between topoisomerase II and a helicase,
possibly SGS-1. If the interpretation is correct, any defect in the
functioning of this segregation machine (helicase or topoisomerase II mutations) would result in chromosome breakage. This
Replication and transcription require that the DNA be accessible
to the enzymatic machineries involved in these processes. It was
suggested, at least for the initiation steps, that nucleosomal structures must be transiently disrupted, then reassembled on newly
replicated DNA or after passage of a transcription complex
(Bonne-Andrea et al., 1990; Adams and Workman, 1993).
Nucleosome disruption may occur via the positive supercoiling produced by a helicase tracking through the DNA
duplex in the presence of a topoisomerase. This idea is based
on the twin supercoiled model imagined by Liu and Wang
(1987) for transcription. In the hypothetical model in Fig. 2C,
the helicase locally separates the DNA strands in front of the
topoisomerase, producing positive supercoils ahead and
negative supercoils behind (Liu and Wang, 1987; Koo et al.,
1991). Positive supercoiling is absorbed by nucleosome disruption, while negative supercoiling is removed by the topoisomerase, allowing the double helix to rewind. This implies
that the topoisomerase acting in this process may selectively
remove negative supercoils (like prokaryotic topo I), or that
positive supercoils are not targeted for immediate removal.
Nucleosome disruption was recently demonstrated by
Ramsperger and Stahl (1995), who showed that purified SV40 T
antigen, acting as a helicase on SV40 replication origin, was able
to displace nucleosomes in vitro. The reaction was only observed
on a DNA with free ends, suggesting that the negative supercoiling produced behind the helicase must be removed, while
positive supercoiling might be absorbed by nucleosome disruption in front of the helicase*. The authors proposed that in vivo,
a modified topoisomerase may remove the negative supercoiling.
As mentioned above, direct binding of T antigen to topoisomerase I was recently demonstrated (Simmons et al., 1996).
The model of nucleosome removal does not, of course, only
apply to replication, but also to repair, transcription, and
recombination. Indeed, helicases appear systematically associated with repair and transcription complexes (Buratowski,
1993; Seroz et al., 1995) and might be used in concert with a
*Addition of topo I or II (both able to relax positive or negative supercoils with the same
efficiency) in the reaction with circular DNA did not permit nucleosome disruption, presumably because both topoisomerases also remove the positive stress necessary for disruption.
When helicase and topoisomerase meet 1347
Segregation of newly
replicated Chromosomes ?
Progression of a
Replication Fork ?
A
AAAAAA
AAAAAA
AAAAAA
AAAAAAAA
AAAAAAAA
AAAAAAAA
T
H
Helicase
matrix
Replication
complex
+
Topoisomerase
Recombination
and genome
stability?
Disruption of
nucleosomal
structure and
transcription ?
B
Topo II
Helicase
DNA supercoiling ?
Fig. 1. Putative cooperation of helicases with topoisomerases in
DNA metabolism.
topoisomerase to disrupt chromatin structure in an appropriate
region. The same model may work in reverse to promote nucleosome assembly, if a topoisomerase removing positive
supercoils works in front of the helicase: then, negative supercoils generated behind the helicase may serve to assemble
nucleosomes, instead of being relaxed by a topoisomerase.
Such a machine may serve as a negative supercoiling force in
eukaryotes and may explain why there is no bacterial-like
DNA gyrase activity in these organisms (see Fig. 3C).
Very recently, Hamiche and Prunell proposed that positive
supercoiling may trigger the flipping of the (H3-H4)2 tetramer
from a left-handed to a right-handed form (Hamiche et al.,
1996). They suggested that the positive supercoiling produced
in front of a transcription complex may be absorbed by this
histone flipping; this mechanism then behaves as a ‘eukaryotic
gyrase’, leaving negative supercoils behind the complex.
DNA SUPERCOILING: THE CASE OF REVERSE
GYRASE FROM HYPERTHERMOPHILES
Reverse gyrase, discovered in hyperthermophilic archaebacteria (Kikuchi and Asai, 1984), has the unique property of being
able to increase the DNA linking number, producing positive
supercoils in a relaxed circular DNA at the expense of ATP
(reviewed by Duguet, 1995).
Mechanistic studies have shown that reverse gyrase transiently cleaves a single DNA strand, forming a covalent link
with the 5′ end, a characteristic of eubacterial topoisomerase I
(Jaxel et al., 1989). How does an ATP-dependent topoisomerase I promote a reaction of supercoiling? A strong clue is
given by sequence data, showing that reverse gyrase is made
of both a helicase-like domain and a topoisomerase I domain
in the same polypeptide (Confalonieri et al., 1993).
Fig. 3A,B describes a plausible mechanism of positive DNA
supercoiling derived from the model of Liu and Wang (1987):
tracking of the helicase domain through the DNA duplex
would produce two waves of supercoiling, positive in front,
and negative behind. Specific relaxation of the negatively
supercoiled region by the associated topoisomerase domain
would produce net positive supercoils. It is noteworthy that the
early models of negative supercoiling by bacterial DNA gyrase
were based on this principle with specific relaxation of positive
supercoils (Gellert et al., 1978) (Fig. 3C).
C
T H
normal helix
Overwinding
(positive supercoiling)
T H
Fig. 2. Interaction between helicases and topoisomerases in DNA
replication and nucleosome disruption. (A) The swivel machine is
represented by the association of a 3′ topoisomerase I (green) with a
helicase (red) anchored on a fixed structure, i.e. the nuclear matrix. 3′
Topo I is the type I topoisomerase usually found in eukaryotes that
binds the 3′ end of the broken DNA strand, and removes positive or
negative supercoils with the same efficiency. As replicating DNA
moves through the structure, the two parental strands (black) are
separated by the helicase, while positive supercoiling is removed by the
3′ topoisomerase. (B) A machine able to separate the daughter
molecules at the end of replication is formed by a helicase (red)
removing the last turns of parental DNA and a type II topoisomerase
(green) untangling the daughter duplexes. (C) Nucleosome disruption.
The positive supercoiling produced by the translocating helicase H (red)
destabilizes the nucleosome, while a topoisomerase T (5′ or 3′ Topo I,
or eukaryotic topo II, green) efficiently relaxes the negative
supercoiling, reforming the normal duplex behind the helicase. 5′ Topo
I is the type I topoisomerase, usually found in prokaryotes, and also
present in yeast. It binds the 5′ end of the broken DNA strand and
exclusively removes negative supercoils. Note that in this figure (and in
Figs 3 and 5), helicases and topoisomerases are represented by rings
around the DNA double-helix. Several recent data on the spatial
structure of these enzymes suggest that they indeed form ring
structures.
Other examples of supercoiling by a helicase-plus-topoisomerase mechanism have been described where helicase and
topoisomerase are not associated in the same polypeptide (Koo
1348 M. Duguet
merase domains. Such functions may also have been conserved
in eukaryotic cells (see below). As first suggested by Kikuchi
(1990), reverse gyrase may be used as a powerful ‘renaturase’
or ‘reformatase’ (see Fig. 5) to eliminate a variety of abnormal
DNA structures such as extruded DNA cruciforms, triple DNA
helices, Z DNA, mismatch regions, or even recombination intermediates. Alternatively, reverse gyrase may help to rapidly
rewind the double helix after passage of a transcription complex
or during the branch migration of recombination junctions. All
these processes must be especially destabilizing at high temperature as the two DNA strands are less tightly associated.
H
A
Overwinding
(positive supercoiling)
Underwinding
(negative supercoiling)
B
5'T
H
Overwinding
(positive supercoiling)
Normal Helix
RECOMBINATION AND GENOME STABILITY
Reverse gyrase
C
H
Underwinding
(negative supercoiling)
3'T
Normal Helix
"Gyrase-like activity"
Fig. 3. DNA supercoiling by the cooperation of a helicase and a
topoisomerase. (A) The translocating helicase (red) produces negative
supercoiling upstream and positive supercoiling downstream. (B) If a
5′ topo I (green) works behind the helicase to specifically relax
negative supercoils, the machine continously increases the DNA
linking number and is a reverse gyrase. (C) If a 3′ topo I (green) works
in front of the helicase to remove positive supercoils, then the machine
decreases the DNA linking number and behaves as a ‘gyrase’.
et al., 1991; Zhang et al., 1990; Quinn et al., 1996). Indeed,
recently, a reverse gyrase with two different subunits has been
isolated from a hyperthermophilic methanogen (Kozyavkin et
al., 1994; Krah et al., 1995).
Finally, the ubiquitous presence of a reverse gyrase activity in
all of the hyperthermophilic organisms so far tested, archaebacterial or eubacterial (Bouthier de la Tour et al., 1991), addresses
the question of its biological function. The simplest idea is that
positive supercoiling prevents DNA from melting at high temperature (Kikuchi and Asai, 1984). However, we can speculate
on other more subtle roles for reverse gyrase, which take
advantage of a cooperation between the helicase and topoiso-
A
The essential role that DNA helicases play in genetic recombination and genome stability has long been recognized. In
contrast, the role that topoisomerases play in these processes still
remains obscure. It was proposed by Wang et al. (1990) that this
role is a ‘double-edged sword’: on the one hand, topoisomerases
may be necessary to promote recombination by their strand-transferase activity and (or) by allowing formation of plectonemically
wound recombination intermediates. On the other hand, topoisomerases may help to repress recombination, via mechanisms that
are not presently understood (Wang et al., 1990).
Possible clues to the role of topoisomerases in recombination
and genome stability recently emerged through their interactions
with helicases. The first clue is provided by a recent report that
a helicase is able to transform a frozen intermediate of topoisomerase II bound to DNA into a permanent double-strand break
in vitro (Howard et al., 1994). This is illustrated in Fig. 4A, where
tracking helicases displace the non-covalently bound 3′ ends,
generating a double-strand break upon dissociation of topoisomerase II subunits. This mechanism may be used in vivo to
provide the DNA breaks necessary to initiate recombination
(Szostak et al., 1983). Indeed, recent analysis of the DS breaks
occurring in yeast meiosis indicate that the 5′ DNA ends are covalently bound to Spo 11 (Keeney et al., 1997), a member of an
atypical topoisomerase II family recently discovered in the
archaea (Bergerat et al., 1997). Since the archaebacterial enzyme,
called Topo VI, presents a classical topoisomerase II activity, an
attractive hypothesis is that in yeast, Spo 11, modified by its interaction with a helicase, is responsible for this cleavage activity.
A second series of observations may explain the dual role of
TOPO II
B
Cleavage
Site 1
Site 2
T H
T H
Heteroduplex
binding to site 1
Helicase
Fig. 4. Putative cooperation between helicases and
topoisomerases in genetic recombination.
(A) Production of a permanent double-strand
break from a topo II cleavable complex by strand
displacement involving a helicase (see Szostak et
al., 1983). (B) The putative role of
helicase/topoisomerase complexes in the
suppression of recombination intermediates or, on
the contrary, in the promotion of branch migration.
Disruption of
recombination
intermediate
Strand displacement
Dissociation
binding to site 2
branch migration
When helicase and topoisomerase meet 1349
H
3'T
Swivelase
migration. Thus, by removing ‘undesired’ heteroduplex
regions as well as various abnormal DNA structures,
helicase/topoisomerase complexes may contribute to genome
stability and (or) promote recombinational exchange.
CONCLUDING REMARKS
TII
H
Segregatase
Daughter
DNA duplexes
Parental strands
5'T
H
Reformatase
AAAAAAA
AAAAAAA
Normal Helix
Abnormal DNA
Structure
Fig. 5. Possible DNA machines by various combinations of
helicase/topoisomerase complexes. Note that: (i) the ‘swivelase’ may
work independently of replication, either in front of a transcription
complex, or as a chromatin assembly machine; (ii) the ‘reformatase’
may also work to rewind the duplex behind the transcription complex.
topoisomerases as activators or repressors of recombination.
Several years ago, a new type I topoisomerase, named Topo III
was discovered in yeast through the hyper-recombination and
slow growth phenotype of its mutants (Wallis et al., 1989).
Remarkably, this eukaryotic topoisomerase belongs in fact to
the prokaryotic topoisomerase I family, since it binds the 5′ ends
of DNA and specifically removes negative supercoils (Kim and
Wang, 1992). A unique suppressor of top3 null mutations was
isolated and turned out to be the putative helicase SGS-1, or
slow growth suppressor (Gangloff et al., 1994) also named TPS1, for topoisomerase supressor (Romeo et al., 1992). SGS-1 was
found to directly interact with topo III in vivo, and also with
topo II, see above. Gangloff and coworkers then proposed that
the SGS-1/Topo III complex acts as a ‘eukaryotic reverse
gyrase’ able to repress recombination by a putative positive
supercoiling activity (see section above and Fig. 3). Such an
activity has not yet been detected. Nevertheless, repression of
recombination by a helicase/topoisomerase complex may occur
in a more direct way without requiring positive DNA supercoiling. Indeed, several observations of an antipairing activity
of a DNA helicase have recently been made (Kodadek, 1991;
Morel et al., 1993; Adams et al., 1994).
Fig. 4B describes how a helicase/topoisomerase machine
may either disrupt unstable recombination intermediates, or
promote branch migration. As pointed out by Wang, recombination synapsis may be seen as a two-way street (Wang et al.,
1990). In path 1, the helicase proceeds through the heteroduplex and disrupts it, while behind the helicase, the topoisomerase catalyzes rewinding of the normal duplex by its strandpassage activity. Conversely, in path 2, the machine binds to
the right end of the heteroduplex and promotes branch
Although most of the ideas presented here remain speculative,
one can imagine that cooperation between helicases and topoisomerases has been exploited again and again in living cells.
Moreover, as suggested in Fig. 5, the various combinations of
helicases and topoisomerases may produce different kinds of
machines working on DNA.
For instance, with a 3′ topoisomerase I placed in front of a
helicase, the machine may work as a ‘swivelase’ acting in DNA
replication, or acting in front of a transcription complex. In the
absence of replication, the machine could also work as a
‘gyrase’, producing net negative supercoiling, or as a
chromatin assembly machine (see also Fig. 3).
With a type II topoisomerase behind the helicase, the
machine may work as a ‘segregatase’, to separate newly replicated daughter chromosomes at the end of replication.
Finally, with a 5′ topoisomerase behind a helicase, the
machine may work as a ‘reformatase’, disrupting chromatin
structure and eliminating all kinds of altered DNA structures,
including cruciforms, Z DNA regions, triple helices, mismatch
or slippage regions in repetitive sequences, or immature recombination intermediates. It may also work as a ‘reverse gyrase’,
producing net positive supercoiling into DNA.
It would be interesting to test these different possibilities, in
particular the disruption of chromatin structure or the elimination of recombination intermediates at least in vitro.
In any case, one can confidently predict that in the future,
greater evidence for the cooperation between helicases and
topoisomerases in the maintenance and stability of the genome
will appear.
I thank my laboratory colleagues for fruitful discussions, and David
E. Adams for critical reading of the manuscript. The Laboratoire
d’Enzymologie des Acides Nucléiques is supported by funds from
CNRS and Université Paris-Sud (URA 1354) and Association pour la
Recherche contre le Cancer (ARC).
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