Minireview
C.1
DNA Topoisomerases: Why So
Many?*
James C. Wang
From the Department of Biochemistry and Molecular
Biology, Harvard University, Cambridge,
Massachusetts 02138
Several new DNA topoisomerases have beendiscovered
recently. In the yeast Saccharomyces cereuisiae, a gene has
been identified to encode a protein homologous to eubacterial
DNA topoisomerase I; this gene has been termed TOP3 and
its product DNA topoisomerase
I11 (1).Sequence datasuggest
that a yeast gene HPRl may encode yet another DNA topoisomerase (2,3). In Escherichiacoli, two genes parC andparE
have been found tocode for a new topoisomerase termed DNA
topoisomerase IV; the amino acid sequences of the parC and
p a r E polypeptides are homologous to, respectively, those of
the A and B subunit of DNA gyrase, also termed bacterial
DNA topoisomerase I1 (4).
These new members of the DNA topoisomerasefamily are
not merely stand-ins or substitutesof their more extensively
studied relatives, because mutations in their structuralgenes
effect distinct phenotypes. Yeasttop3 mutants are characterized by poor growth and higher frequency of recombination
between a class of repetitive sequences termed 6 sequences
(1); E. coliparC temperature-sensitive mutants areknown (5)
and parCor parE mutants exhibit defects in thesegregation
of newly replicated chromosomes (4,5). It is the functional
distinctiveness of these new enzymes that leads to the question in the title of this review, why are there so many DNA
topoisomerases?
Supercoiling and Relaxationof Intracellular DNA
Two decades ago, when the first DNA topoisomerase was
discovered in extracts of E. coli cells (6), DNA replication
topped the list of potential biological processes that might
require such a n enzyme. The separation of DNA strands in
one region should lead to overwinding of the DNA in other
regions; the bacterial enzyme was shown, however, to relax
preferentially underwound or
negatively supercoiledDNA (6).
In 1976, a second DNA topoisomerase was discovered in E.
coli (7). This enzyme, termed DNA gyrase or bacterial DNA
topoisomerase 11, is both a DNA topoisomerase anda DNAdependent ATPase, and itcouples ATP hydrolysis and DNA
negative supercoiling. The enzymatic properties of DNA gyrase immediately led to a number of suggestions regarding its
physiological roles. First, unlikeE. coli DNA topoisomerase I,
gyrase is capable of removing positive supercoils in the presence of ATP and thus appears to be
more suitable in solving
the problem of DNA overwinding during semiconservative
replication. Second,the negativesupercoiling of DNA by
DNA gyrase hints strongly that DNA
supercoiling might occur
inside a living cell. Furthermore, because the actionsof DNA
gyrase and DNA topoisomerase I are diametric, they might
*The bulk of the work on DNA topoisomerases in the author's
laboratory has been supported by grants from the National Institutes
of Health (GM24544 and its predecessor GM14621; CA47958).
THEJOURNALO F BIOLOGICAL CHEMISTRY
Vol. 266, No. 11, Issue of April 15, pp. 6659-6662, 1991
1991 hy The American Society for Biochemistry and Molecular Biology, Inc.
Printed in 1J.S.A.
form an opposing pairinthe
regulation of the degree of
supercoiling of intracellular DNA (8-10).
The idea that DNA gyrase and DNA topoisomerase I regulate DNA supercoiling in uiuo is supported by genetic evidence that the lethal phenotype of E. coli topA null mutants
is compensated by mutations in gyrA or gyrB that reduce
gyrase activity (11-13). Several experimental findings could
not be accounted for, however, by the model. In 1983, Lockshon and Morris(14) found that inhibitionof DNA gyrase in
E. coli harboring pBR322 resulted in the accumulation of
positivesupercoils in the plasmid. Itmadenosense
why
inhibiting a negative supercoiling activity should lead topositive supercoiling. Pruss (15) also made an interesting finding
in 1985 that pBR322 isolated from E. colitopA mutants is
about twice as negatively supercoiled as the same plasmid
from topA+ strains, but samples of a derivative of pBR322
isolated from topA- and topA' strains show little difference
in supercoiling. Subsequently, Pruss and Drlica (16) showed
that thedifference in supercoiling of pBR322 or its derivatives
in topA- and topA+ strainsisstronglydependentonthe
transcription of the region of the plasmidencoding resistance
to thedrug tetracycline. Why should degree
the of supercoiling
of a plasmid depend on theexpression of a gene?
The possibility that transcription mightlead to the supercoiling of the DNA template under specific conditions was
first raised in 1985 (17) prior to the experimentalfindings of
Pruss and Drlica (16). In1987, Liu and Wang (18)proposed
a more general model to account for the resultsof Lockshon
and Morris(14) as well as thoseof Pruss andDrlica (16). The
essence of the model is illustrated in Fig. 1. According to this
model, two oppositely supercoiled domains may accompany
transcription or other processes involving the tracking of a
macromolecular assembly along a DNA. The degree of supercoiling of a particular region of intracellular DNA is determined by how fast the supercoils are being generated by the
tracking process, how fast they can be removed by the DNA
topoisomerases, and how fast the oppositely supercoiled domains can neutralize each other through
diffusional pathways
(18).The last factor isprobably important in the compensation of topA mutations by gyrA or gyrB mutationsin E. coli.
The twin supercoiled domain model of transcription provides a rationale for the existenceof two seemingly diametric
DNA topoisomerases in eubacteria, DNA gyrase and topoiof each other,
somerase I. Rather than countering the actions
these two enzymes actually act jointly torelax the two oppositely supercoiled domains that may form during transcription, or more generally in processes involving the trackingof
a macromolecular assembly along a DNA. Each of the two
topoisomerases hasevolved beautifully to accomplish its mission. The specificity of eubacterial DNA topoisomerase I for
the negatively supercoiled domain is achieved byits unpairing
enzyme-DNA
of a short stretch of DNA in forming the active
complex (6, 19, 20); in contrast, the right-handedwrapping of
a DNA segment around DNA gyrase (21) causes the enzyme
to preferentially bind to the
positively supercoiled domain. In
cells undergoing active transcription, gyrase might act as an
enzyme that specifically removes positive supercoils
rather
than as one that introducesnegative supercoils (18).
This division of labor between the two bacterialtopoisomerases is, of course, not the only way of relaxing oppo-
6659
0
Minireview: DNA Topoisomerases
6660
\iR
(A)
(6)
the major activity thatrelaxes negatively supercoiled DNA i n
vivo. Furthermore, because of the accumulation of positive
supercoilsin plasmidsuponinhibition
of DNA gyrase by
Novobiocin (14, 26), gyrase appears to be the only enzyme
capable of relaxing positive supercoils unless the otherrelaxation activities in E. coli are also inhibited by this drug. If
DNA topoisomerases I and I1 in yeast, and DNA topoisomerase I and DNA gyrase in E. coli are the dominant activities
in the relaxationof supercoiled domains i n viuo, what are the
functions of the other DNAtopoisomerases?
FIG. 1. A : top, a transcriptional ensemble R is shown on a DNA
DNA Topoisomerasesand the Unraveling of
segment, the ends of which-are anchored to a certain cellular entity
Intertwined DNA Strands or Duplex DNA Pairs
E. As R tracks along the
DNA, it would be expected toencircle around
the templatebecause of the helical geometryof DNA. If this encircling
As mentioned earlier in this review, in terms of the physmotion is prevented, through anchoring of any component of R to a
iological
roles of DNA topoisomerases the
well known “swivel”
cellular entity or the DNA itself, or because of a high viscous drag
accompanying the movementof R in a cellular milieu, then the DNA problemfor the semiconservative replication of two interin theyears.
must turn around itshelical axis instead. Rotationof the DNAwould twined DNA chains dominated the thinking early
in turn generate
positive supercoilsahead of R and negative supercoils From the molecular point of view, there are actually two
behind it.Bottom, the neteffect of preventing the turning
of R around distinct topological problems in the replicationof DNA. The
the DNA is to squeeze all helical turns ahead of R into a shorter and first accompanies the elongation stage of DNA replication
shorter region as R advances, and the opposite occurs hehind R. B,
the anchoringof the endsof the DNA segment toE, depicted inA , is and can be viewed as a special case of the twin supercoiled
not an essential featureof the model. Here the terminal elements are domain model involving the tracking of a macromolecular
assembly along a DNA. As the replication forkadvances,
combined to illustrate the situation for a plasmid E could he nonexistent, in which case the viscous drag against the rotation of the positive supercoils are generated aheadof it. Behind thefork,
DNA itself between the oppositely supercoileddomains would provide the separated parental strands can be viewed as a limiting
the only retarding force against the merging of these domains; in the case of negative supercoiling; separated strands represent the
other extreme, E could he an immobile site on the cell membrane.
highest degree of negative supercoiling achievable in a duplex
Drawings are adopted from Refs. 18 and 26.
sitely supercoiled domains. In eukaryotes, the entrance of a
new enzyme and the evolution of an old one have demonstrated clearly that it ispossible for one enzyme to relax both
supercoiled domains. Eukaryotic DNA topoisomerase
I, which
shows little sequence homology to either eubacterial DNA
topoisomerase I or DNA gyrase and thus represents a new
family of the topoisomerase clan, is well known for its relaxation of negatively and positively supercoiled domains with
nearly equal efficiency (22); eukaryotic DNA topoisomerase
11, which is homologous t o bacterial DNA gyrase, similarly
relaxes both oppositely supercoiled domains (23). While the
bacterial type I1 enzyme binds preferentially to a positively
rather than negatively supercoiled region due to the righthanded wrapping of a DNA segment around theenzyme, this
DNA wrap is absent in the DNA-eukaryotic type I1 enzyme
complex (23); probably as a consequence, the eukaryotic enzyme binds to oppositelysupercoiled domainswithoutan
intrinsic bias.
DNA. In E. coli, as described earlier, DNA gyrase would be
the best candidate tofulfill the role of removing the positive
supercoils, in agreement with experimental findings (14, 26).
In yeast, either DNA topoisomeraseI or I1 should do, which
is again supportedby experimental data (27).
The second problem occurs near the endof the elongation
stepandisillustratedin
Fig. 2 fora pair of converging
replication forks. Depending on the
relative rate of unraveling
/
Path A
\
Path B
Are All DNA Topoisomerases Involved in the
Relaxation of Supercoiled Domains in Vivo?
Giaever and Wang (24) argue that in eukaryotes, DNA
topoisomerases I and I1 are the only activities that can relax
DNA Synth.
supercoiled intracellularDNA efficiently. Intheirexperi6 Ligation
ments, an endogenous yeast plasmid, termed the2r( plasmid,
wasshown to become positivelysupercoiledonly
in cells
expressing E. coli DNA topoisomerase I and only when both
yeast DNA topoisomerases I and I1 were inactivated. If there
+
is a third yeast enzyme capableof relaxing positive supercoils
/
efficiently, there should be no accumulation
of positive supercoils in those experiments; if there is a third yeast enzyme
capable of relaxing negative supercoils efficiently, the accumulation of positivesupercoils in the plasmid should not
depend on theexpression of the E. coli enzyme.
In E. coli, Bliska and Cozzarelli (25) showed that intracelFully
Replicated
&
Segregated
Progenies
lular plasmids become less negatively supercoiled upon inhibition of DNA gyrase by Norfloxacin in a topA+ but not in a
FIG. 2. TWOpaths for the merging of a pair of converging
topA- strain. Thus it appears that DNA topoisomerase I is replication forks. This drawingwas modified from Ref. 43.
Minireview: DNA Topoisomerases
the intertwined parental strands, which requires a DNA topoisomerase, and that for the completion of progeny strand
synthesis, whichinvolves the polymerizing machinery and
DNA ligase, two extreme events may occur; in Path A, unraveling is slow and the replicating DNA ends up as a pair of
multiply intertwined duplex molecules (Ref. 43; catenanes if
the original DNA is in the form of a ring); in Path B,
completion of progeny strand synthesis is slow, and a pair of
gapped but unlinked progeny molecules is formed.
From theknown enzymatic propertiesof type I and type I1
DNA topoisomerases, a type I1 enzyme is needed to unlink
the intertwined duplex molecules that are formed in Path A.
In the yeasts S. cerevisiae and Schizosaccharomyces pombe,
Path A is apparently a majorroute near the end
of replication,
and the essentiality
of DNA topoisomerase I1 rests mostlikely
on its role in the unlinking of the intertwined duplex molecules (28). In E. coli, the Faror partition-defective phenotype,
namely the formation of large nucleoids in the midcell, has
been observed for both gyrase and DNA topoisomerase IV
mutants, suggesting that both type I1 DNA topoisomerases
are necessary for this step (4, 5 , 29). Mutations in the E. coli
minB locus have also been shown to affect nucleoid segregation and plasmid supercoiling, and the possibility that the
minB products might be a topoisomerase or interacting with
atopoisomeraseinvolved
in chromosomal partitioning has
been raised (30). Why more than one type I1 DNA topoisomerase is needed for chromosomal segregation
is unclear. In
addition to binding to DNA, E. coli DNA topoisomerase IV
has also been implicated to interact with the cell membrane
( 4 , 5 ) . The ways the various topoisomerases interact with the
cell membrane or other cellular entities might underlie the
requirement of multiple topoisomerases in thesegregation of
chromosomes.
Path A is unlikely to be the only path, however, and Path
B provides an alternate route forsegregating the pairs of
progeny molecules (for examples, see Refs. 31-33). In Path
B, either a type I or a type I1 DNA topoisomerase may unlink
the intertwined structure. E. coli DNA topoisomerase I, for
example, can link or unlink DNA containing single-stranded
regions efficiently (9, 22). E. coli DNA topoisomerase 111, as
well as E. coli DNA topoisomerase I,has been shownto
efficiently unlink gapped plasmid DNA near the end of a
round of replication in vitro (31, 32).
It should be emphasized that although PathsA and B are
parallel pathways, the use of type I DNA topoisomerases in
Path B cannot salvage the lethal effect of blocking Path A
through the inactivation of the type I1 DNA topoisomerases.
This follows from chromosomal loss or breakage, which would
occur if the intertwined DNA intermediate depicted in Path
A is not unlinked before the partition of the chromosomes.
In E. coli, it has been suggested that DNA topoisomerases
I and I11 may participate in Path B (31, 32). Similarly, the
poor growth phenotype of yeast DNA topoisomerase I11 might
be related to itsrole in such a path (1, 3).Inactivation of the
type I enzymes does not necessarily lead to cell killing, however, because the type I1 enzymes can substitute for them in
A.
this pathor provide an alternative path, namely Path
6661
to suppress recombination within thecluster. Kim and Wang
(35) foundthat in yeast Atop1 top2 ts cells grown at a
permissive temperature, the rDNA gene cluster is unstable
and excision of the genes occurs to form extracellular rDNA
rings; expression of either DNA topoisomerase I or increasing
the level of DNA topoisomerase I1 in this strain restores the
stability of the chromosomal rDNA cluster, and the extrachromosomal rings are shown to reintegrate into their usual
locus on the chromosome. As mentioned earlier, null mutaTOP3 gene increase the frequency of recomtions in the yeast
bination between the 6 sequences (1).
Although it has been suggested that theincrease in recombination frequency in top mutants might be a result of these
mutations on DNA supercoiling, this interpretation is only
straightforward for the Atop1 top2 tsdouble mutant results of
Kim and Wang(35); the strain theyused is completely devoid
of DNA topoisomerase I, and even at a permissive temperature there is only a low level of DNA topoisomerase 11. Thus,
neither of the twomajorsupercoil
relaxationactivities is
present a t adequate levels under their experimental conditions, and template supercoiling in the actively transcribed
rDNA gene cluster may verywell occur. The resultsof Christman et al. (34) with the topl cells can be fitted into the same
scheme, asDNA topoisomerase I is the most potent relaxation
activity in eukaryoticcells. Their results with TOPl+ top2 ts
cells cannot be readily explained in the same way, however,
as the presenceof DNA topoisomerase I should be sufficient
to solve the supercoiling problem. Because the experiments
with the TOPl+ top2 ts strain were carried out at semipermissive temperatures, these results might be more complitopl TOP2' cells, and the
cated than those obtained with
molecular mechanisms underlying the increase in recombination in thesetwo cases might not be the same.
The effects of TOP3 mutations on recombination in particular are difficult to account for by the supercoiling explanation. I n vivo and in vitro, the TOP3 gene product is at best a
rather weak relaxation activity. One could invoke DNA sequence specificity to argue that
DNA topoisomerase I11 might
be a better relaxation activityfor certain DNA sequences, but
this argumentdoes not circumvent the
difficulty of explaining
the top3 mutant phenotypes in the presence of other strong
relaxation activities, namelyDNA topoisomerases I and 11. It
is this dilemma that
led Wang et al. (3) to postulate that there
might be cellularprocesses that would normallyminimize
mitotic recombination by resolving inadventitiously paired
DNA strands. Conceivably, DNA topoisomerases that are
inefficient in relaxingsupercoiledregions
but efficientin
unlinking intertwined strands, such as yeast or E. coli DNA
topoisomerase 111, might be participating in these processes.
DNA Topoisomerases and Chromosomal Folding
Those who have used a long garden hose would appreciate
the problem of coiling such an object into an ordered form.
Even in the early years of DNA topoisomerase studies, the
plausible involvement of such an enzyme in chromosomal
condensation and decondensation was recognized (36). Recently, genetic and cytological studies withS. pombe (37),and
in vitro studies with cell extracts (38) have implicated a role
DNA Topoisomerases andGenome Stability
of eukaryotic DNA topoisomerase I1 in chromosomal condenAn exciting recent finding in the study of DNA topoiso- sation and decondensation.
merases is their involvement in the maintenance of genome
From the pointof view of DNA topology, either eukaryotic
stability.Christman et al. (34) foundthatyeast topl null DNA topoisomerase I or I1 should be sufficient to overcome
mutants or top2 ts mutants at semipermissive temperatures the problems of twisting and coiling of a long DNA into an
exhibit a much higherfrequency of recombination in the
(DNA topoisomerase I would
organized and compact structure
wild-type controls and not be able to substitute for DNA topoisomerase I1 only if
ribosomal DNA gene cluster than their
suggested that bothDNA topoisomerases I and I1 are required interlocking of the DNA loops in the compact structure is
6662
Minireview: DNA Topoisomerases
necessary). One explanation of the DNA topoisomerase I1 stranded DNA as the genetic material has set the stage for
requirement in the presence of DNA topoisomerase I is a their entrance.
structural role of the type I1 enzyme itself in the organization
Acknowledgment-I thank all m y p r e s e n t and past co-workers for
of the condensed chromosome. Immunomicroscopy has imm a k i n g the study of DNA topoisomerases a joyful undertaking.
plicated eukaryotic DNA topoisomerase I1 as a major component of the chromosome scaffold (39-42).
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Conclusion
One could view the ascendencyof the DNA topoisomerases
in nature as
a consequenceof the selection of double-stranded
DNA as the genetic material. A plethora of problems accompanying the various transactionsinvolving DNA is all deeply
rooted in the bihelicalgeometry of thismaster molecule.
Tracking of amacromolecularassembly
along a DNA, in
transcription or replication for example, may generate supercoiled domains that must be attended to. The unraveling of
the complementary DNA strands near the endof a round of
replication, and the formation
of multiply intertwineddoublestranded progeny molecules that may occur during thisprocess, pose unique topological problems. Folding of a chromatin
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solved. The list cango on.
All the problems cited above would have disappeared if
DNA existed as short linear
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DNA topoisomerases, through their manipulation of DNA
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for example, hasprofound effects on DNA structureand
interactions between DNA and other molecules; there is also
ample evidence that DNA supercoiling affects many cellular
processes.
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magicians; they open andclose gates inDNA without leaving
a trace, and they enable
two DNA strands or duplexes to pass
each other asif the physical laws of spatial exclusion do not
exist. Because the biological functions of the DNA topoisomerases are deeply rooted in the double helix structure of
DNA, it should notbe surprising that theseenzymes participate in nearly all biological processesinvolving DNA the
recent discovery of several new DNA topoisomeraseshas
brought a deeper understanding of their many vital roles in
living cells. Why are there so many DNA topoisomerases?
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