R778 Dispatch

DNA topology: Topoisomerases keep it simple

Andrew D. Bates* and Anthony Maxwell†

The ability of type II DNA topoisomerases to perturb the DNA gyrase, which is the only type II enzyme known to
equilibrium distributions of DNA topoisomers is a be capable of introducing supercoils into DNA, the
consequence of their ability to hydrolyse ATP. A sliding requirement for ATP is clear. The introduction of super-
mechanism of topoisomerase action has been coils into a relaxed DNA circle is energetically
proposed to account for this phenomenon. unfavourable and it seems that, under the right condi-
tions, much of the energy of ATP hydrolysis can be con-
Addresses: *School of Biological Sciences, University of Liverpool, Life
Sciences Building, Crown Street, Liverpool L69 7ZB, UK. E-mail:
verted into DNA supercoiling energy [4–6]. Gyrase
[email protected]. †Department of Biochemistry, University of Leicester, performs this directional intramolecular reaction by
University Road, Leicester, LE1 7RH, UK. E-mail: [email protected]. wrapping a segment of DNA around itself, and deliver-
ing a nearby T segment to the ATP-operated clamp with
Current Biology 1997, 7:R778–R781
http://biomednet.com/elecref/09609822007R0778 a specific orientation [7].

© Current Biology Ltd ISSN 0960-9822 In the case of the eukaryotic type II enzymes or the
bacterial enzyme topoisomerase IV (topo IV), which are
One of the basic tenets of enzyme-catalysed reactions is specifically implicated in the unlinking of daughter
that an enzyme cannot alter the equilibrium between chromosomes after DNA replication [8,9], the energy
substrate and product — it can only accelerate the rate of requirement is not so clear. These enzymes do not wrap
interconversion between them. However, if the enzyme DNA, and they catalyse reactions that appear to be ener-
couples an energy-yielding reaction, such as the hydrolysis getically favourable, such as the relaxation of super-
of ATP, to the conversion of substrate to product, then an coiled DNA or the unlinking of catenanes, yet they too
apparently non-equilibrium mixture can be generated. It require the hydrolysis of ATP. It has been suggested
has long been a puzzle why many type II DNA that this energy input is needed to drive the enzyme
topoisomerases hydrolyse ATP while catalysing energeti- through an ordered series of conformational changes
cally favourable reactions, such as the relaxation of [10], in other words, to facilitate the opening and closing
supercoiled DNA. Recent experiments by Rybenkov et al.
[1] show that these enzymes are able to reduce the
topological complexity of DNA relative to equilibrium Figure 1
values, and ATP hydrolysis is invoked to explain their
ability to perturb the thermodynamic equilibrium.
(a)
DNA topoisomerases catalyse the interconversion of differ-
ent topological forms of DNA, including relaxed and super-
coiled, knotted and unknotted, and catenated (linked) and
unlinked DNA circles (Figure 1). They are classified into
two types: type I enzymes catalyse reactions involving tran-
sient single-strand breaks in DNA, and type II enzymes (b)
catalyse reactions involving transient double-strand breaks
[2]. The current mechanistic model for type II enzymes +
involves the binding of two segments of DNA: a G (gate)
segment, which is broken in both strands by the enzyme
with the formation of covalent bonds between active-site
(c)
tyrosines and 5′ phosphates in the DNA, and a T (trans-
ported) segment, which is captured by an ATP-operated
clamp and passed through the enzyme-stabilised break in
the G segment [3]. This passage of one double-stranded
segment through another, in either an intramolecular or Current Biology
intermolecular fashion, can accomplish all the reactions
illustrated in Figure 1. The reactions carried out by type II topoisomerases: (a) supercoiling/
relaxation; (b) catenation/decatenation; (c) knotting/unknotting. All these
transformations can be performed by the passage of one double-
Most reactions catalysed by type II enzymes require stranded DNA segment through another (double-headed arrows).
ATP hydrolysis. In the case of the bacterial enzyme
 Dispatch R779

Figure 2 using an analogous method, and similarly found to be far

from the equilibrium value when enzyme and ATP were
present: 90-fold lower for pAB4, and 50-fold lower for P4.

Equilibrated With supercoiled pAB4 plasmid, the relaxation reaction

mixture was studied and topo IV was found to generate a distribu-
Topo IV + ATP
tion of topoisomers with a narrower range of linking
numbers (Lk) than that generated by wheat-germ topoiso-
Topo IV + ATP merase I, an ATP-independent DNA relaxing enzyme.
This means that topo IV can reduce the level of supercoil-
ing in a sample below the equilibrium level established by
Steady-state thermal energy [14]. Similar experiments were carried out
mixture with other type II enzymes from phage T2, Saccharomyces
Topo IV + ATP (Topo IV + ATP) cerevisiae, Drosophila melanogaster and human cells, and
although the magnitude of the effect varied from enzyme
Current Biology
to enzyme, all reduced the fraction of catenanes and knots
A schematic illustration of the reaction of topoisomerase IV with DNA and narrowed the Lk distribution. In other words, all these
catenanes. The steady-state mixture of catenated and unlinked enzymes actively simplify DNA topology relative to the
products formed in the presence of topo IV and ATP contains a lower complexity expected at equilibrium.
proportion of catenanes than the equilibrium mixture formed by the
cyclisation of P4 DNA in the presence of pAB4 plasmid. Catenanes
are in red, and unlinked DNA circles are in green. To comply with the laws of thermodynamics, the energy
of ATP hydrolysis must be invoked to drive these reac-
tions away from their equilibrium positions. With hind-
sight, it seems clear that coupled ATP hydrolysis should
of the ATP-operated clamp that captures the T segment enable type II topoisomerases to generate non-equili-
and initiates the strand-passage process. brated products, given the persuasive example of the
supercoiling reaction catalysed by DNA gyrase (see
A number of recent studies have suggested that these above). The extreme nature of the gyrase case may have
enzymes may be acting more specifically than previously blinded us to the rather more subtle effects of the other
thought — for example topo IV has been shown to be more type II enzymes. The experiments of Rybenkov et al. [1]
active in decatenation than in relaxation reactions [11,12]. elegantly demonstrate these effects, and show that all type
Recent work by Rybenkov et al. [1] now suggests an alter- II enzymes act with a specific directionality: gyrase in the
native explanation for the ATP requirement of these direction of increasing supercoiling complexity, and the
enzymes. In previous studies, these workers developed a other enzymes in the direction of simplifying DNA topol-
system that allows equilibration of catenanes without the ogy. This behaviour is consistent with our understanding
requirement for enzymic catalysis, consisting of a nicked of the roles of these enzymes in vivo; gyrase maintains the
plasmid (pAB4) and linear phage P4 DNA, which has long level of negative supercoiling in bacterial cells and specifi-
cohesive ends [13]. Under conditions of equilibration, the cally removes positive supercoiling generated by transcrip-
P4 DNA molecules circularise, trapping some of the pAB4 tion and replication, whereas the other type II enzymes
plasmids to form dimeric catenanes. The proportion of relax both positive and negative supercoiling, and unlink
catenated DNA at equilibrium can be determined using an daughter chromosomes during and after replication [15].
agarose gel. This system enabled Rybenkov et al. [1] to
compare samples of catenated DNA generated either by To envisage a mechanism that will explain this behaviour
equilibration or by the action of type II topoisomerases is a more difficult matter. In order to perturb the equilib-
(Figure 2). The fractions of catenated DNA formed were rium between catenated and unlinked DNA, for example,
shown to be very different in these two cases. the enzymes need to identify specifically a G segment and
a T segment that are located on two catenated circles in
When a sample with twice the equilibrium concentration preference to a pair of segments on unlinked molecules.
of catenanes was treated with topo IV and ATP at a range Rybenkov et al. [1] propose a model in which each enzyme
of enzyme:DNA ratios, the steady-state fraction of cate- can bind to three sites on DNA. Initial binding to two seg-
nanes in the sample was as much as 16-fold lower than the ments of a DNA circle, the G segment (which will become
value at equilibrium in the absence of enzyme, and this cleaved) and another distant site, might constrain a cate-
same fraction was obtained when the initial substrate was nated or knotted strand or a supercoiled region (destined
a mixture of unlinked circles at the same concentrations to become the T segment) within a shorter loop of the
(Figure 2). For both pAB4 and P4 DNA, the fraction of circle, and thus make capture of the T segment more
knotted versus unknotted species was also measured probable (Figure 3). Although there is some evidence for
R780 Current Biology, Vol 7 No 12

the interaction of yeast topoisomerase II with two distant Figure 3

sites on DNA [16], such an effect will not, of itself, explain
the perturbation of the equilibrium. It is therefore envis-
aged that the enzyme can slide along the DNA at the third G segment
site, constraining the prospective T segment in a progres-
sively smaller region and making its capture increasingly T segment
probable (Figure 3). This directional sliding would have to
be the energy-requiring step of the process.
Topoisomerase

This idea, however, is at odds with what we know of the ATP hydrolysis
properties of the ATP binding and hydrolysing domains of
these enzymes. There is abundant evidence from studies
of gyrase and yeast topoisomerase II that binding of ATP
acts to close a clamp by domain dimerisation, trapping the
T segment, which is then passed through the G segment
[3,17,18]. Subsequent hydrolysis of the ATP re-opens the
clamp ready for a new reaction. It is difficult to imagine a
dual role for ATP hydrolysis both in clamp opening and
closing, and in driving the sliding of the topoisomerase on Current Biology

DNA, but as there is no obvious alternative mechanism to The model proposed by Rybenkov et al. [1] for the ATP-driven
account for these phenomena, we are left with this intrigu- decatenation reaction of type II topoisomerases. The enzyme (blue)
ing model. binds to the G segment and another distant site, at which ATP
hydrolysis can drive the enzyme along the DNA, constraining the T
segment in a smaller loop and making its capture more probable. The
The work of Rybenkov et al. [1] also raises other interest- notch in the enzyme indicates the ATP-operated clamp and the arrows
ing questions. For example, which part of the enzyme indicate the ATP-dependent movement of DNA.
binds the postulated third DNA segment? It would have
been tempting to propose that this segment binds to the
carboxy-terminal domain, which does not have an identi- DNA topological forms in the direction consistent with
fied role in the reaction mechanism of non-gyrase type II their biological role. How the enzymes achieve this
enzymes. However, Rybenkov et al. [1] show that a remains to be shown. It is clear that there are many more
mutant form of yeast topoisomerase II that lacks the last complexities of these remarkable enzymes which remain
approximately 200 amino acids can also perturb the topo- to be unravelled.
logical equilibria (actually in a more extreme fashion than
the full-length enzyme). Acknowledgements
We would like to thank Tim Hammonds, Sotirios Kampranis, Stephen
Robertson and Melisa Wall for helpful discussions and comments on the
Does DNA gyrase have the same ability to generate non- manuscript. AM is a Lister-Jenner Research Fellow.
equilibrium topoisomer products? Although the answer is
obviously ‘yes’ in the case of the supercoiling reaction, it
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 Dispatch R781

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