DNA Topoisomerases

Biochemistry 201
Advanced Molecular Biology
January 7, 2000
Doug Brutlag
Prokaryotes maintain their DNA in a supercoiled state by the combined
action of two enzymes that can alter the linking number of DNA without changing
its primary structure. These enzymes are called DNA topoisomerases. One of the
bacteria enzymes, DNA gyrase, utilizes the energy of ATP to induce supertwists in
the bacterial chromosome and the other, DNA topoisomerase I, relaxes supercoiled
DNA. It is the combined action of these two opposing enzymes that allows bacteria
to maintain a delicate balance and proper superhelical density of the chromosome
required for replication, repair, recombination and gene function.
In higher organisms, supercoiled DNA results from the wrapping and
unwrapping of the DNA in a left-handed helical path around the major structural
proteins of the chromosome, histones. The wrapping of DNA around histones
requires the action of DNA topoisomerases to resolve the topological constraints
imposed during wrapping. When the DNA unwraps from chromatin, the DNA
contains residual negative superhelical turns. It is interesting that higher and lower
organisms appear to have such different mechanism for supertwisting their DNAs.
After discussing how chromosomes fold, we will see that the two mechanisms are
conceptually quite similar.
In addition to the maintenance of the superhelical state of the DNA in
prokaryotes and facilitating chromosome formation in eukaryotes, mutant studies
have given insights into further biological functions of DNA topoisomerases. Yeast
mutants, defective in DNA topoisomerase II, are defective in chromosome
segregation.
Isolation and Mechanism of DNA Topoisomerase I
The first DNA topoisomerase was isolated in 1971 by Jim Wang from E. coli.
He detected an enzymatic activity that caused a supercoiled DNA molecule to
become relaxed but maintained the DNA in a covalently closed form.
The purified enzyme, now called DNA topoisomerase I, required no cofactors
other than divalent metal ions for activity. It works by a mechanism in which one
strand of the DNA helix was nicked and the enzyme became covalently bound to
the 5' phosphate terminus at a nick in a high-energy bond. The intact strand of the
DNA then passes through the nick and the enzyme would then reform the
phosphodiester bond using the energy stored in the protein-DNA linkage.

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DNA Topoisomerases
The hypothetical
intermediate consisting of
DNA topoisomerase I
covalently bound to the 5'
phosphoryl group at a nick in
DNA initially proved elusive.
Conditions that denatured
the enzyme-DNA complex
generated such linkages.
Chemical analysis of the
DNA-protein link showed
that a 5' phosphate from a
phosphodiester bond in DNA
is transesterified to a tyrosine
on the protein.
DNA topoisomerase I
catalyzes many reactions
suggesting it permits one
strand of DNA to physically
pass through a nicked strand.
The most dramatic evidence of
this is that DNA
topoisomerase I will catalyze
the complete renaturation of
two separate complementary
circular DNAs. In this reaction
the DNA topoisomerase I
enzyme catalyzes the change
in the linking number from 0
to the number of DNA twists
in a complete duplex.
Another interesting
reaction that indicates a single
strand breakage and rejoining
is the conversion of single
stranded circles to knotted
forms.
The most disappointing
property of the DNA
topoisomerase I is the
specificity it shows for
negatively supercoiled DNA molecules. DNA topoisomerase I is only capable of
relaxing negatively supercoiled DNAs. The enzyme does not relax DNA molecules
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DNA Topoisomerases
that are overwound (twisted in the positive sense). This means that the enzyme
does not act like a free swivel, but more like a ratchet only allowing rotation of the
nicked strand in one direction about the intact strand.

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DNA Topoisomerases
The ability to release only negative superhelical turns is a severe limitation
because most physiological processes that involve the metabolism of DNA cause
DNA to become overwound! During DNA replication, for example, the two
parental DNA strands separate at the growing fork and this separation induces
positive turns in the parental DNA helix. During transcription, the DNA template
becomes locally unwound in a region near the RNA polymerase but this process
two induces positive turns in the rest of the chromosome.
Eukaryotic DNA Topoisomerase I
DNA topoisomerase I enzymes that use the nicking-closing mechanism have
been isolated from eukaryotic sources as well and they have been found to have a
broader specificity with respect to the types of DNA they will relax.
Most of these enzymes have no requirement for cofactors, neither for energy,
nor for divalent metal ions.
Their mechanism is similar to the nicking-closing mechanism worked out
for DNA topoisomerase I from E. coli except they become covalently attached at the
3' phosphoryl at a nick in DNA and leave a free 5' hydroxyl group.
Eukaryotic nicking-closing enzymes will relax both negative and positive
superhelical turns. They also show no preference for highly supertwisted DNA
versus more relaxed DNA.

Despite these differences in mechanism and specificity, the yeast DNA

topoisomerase I has been shown to effectively substitute for a deficient DNA
topoisomerase I in bacteria.

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DNA Topoisomerases
Structure of human DNA topoisomerase I
Last year the structure of DNA topoisomerase I from Escherichia coli was
determined by Redinbo et al, The crystal structures at 2.1 and 2.5 angstrom
resolution of reconstituted human topoisomerase I comprising the core and
carboxyl-terminal domains in covalent and noncovalent complexes with 22-base
pair DNA duplexes reveal an enzyme that "clamps" around essentially B-form
DNA. The core domain and the first eight residues of the carboxyl-terminal domain
of the enzyme, including the active-site nucleophile tyrosine-723, share significant
structural similarity with the bacteriophage family of DNA integrases. A binding
mode for the anticancer
drug camptothecin is
proposed on the basis of
chemical and
biochemical
information combined
with these threedimensional structures
of topoisomerase IDNA complexes. The
central pore of the
molecule varies from 15
to 20 in diameter and
provides an extensive,
highly positively
charged binding region
for the substrate DNA.
The overall dimensions
of the reconstituted enzyme are 70 by 60 by 60 . The cap of the molecule and
core subdomain III contact one another at two lips (residues 367 to 369 of subdomain
I and residues 497 to 499 of subdomain III), as shown.

DNA Gyrase
The second prokaryotic DNA topoisomerase to be discovered was DNA
gyrase, an enzyme that can actually introduce negative twists into DNA at the
expense of ATP.
DNA gyrase induces negative superhelical turns into a covalently closed
circular DNA at the expense of ATP. Since this reaction causes supercoiling of the
DNA and results in a marked decrease in entropy of the DNA it is unfavorable and
would be expected to require energy.

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DNA Topoisomerases

However,
experiments
indicate that DNA
gyrase can
introduce a limited
number of
supertwists in
DNA (1-2 twists
per molecule of
DNA gyrase) in the
presence of a nonhydrolyzable
analog of ATP.
This reaction
suggests that the
cleavage of ATP to
ADP and
phosphate is
required primarily
for the enzyme to
turn over, or to
recycle.
The enzyme
DNA gyrase
contains four
subunits and is of the
composition A2 B2
The A subunit is
105,000 daltons and
the B subunit is
95,000 daltons
The A subunit
contains the site for
sensitivity to the
antibiotic nalidixic
acid while the B subunit contains the site for sensitivity to novobiocin. Since these
antibiotics have been know to inhibit bacterial DNA replication this implicates an in
important role for DNA gyrase in replication.
Subunit A contains the site of DNA gyrase that binds to DNA and is
responsible for the induction of superhelical turns. Subunit B contains the site for
ATP binding and hydrolysis since inhibition of gyrase by novobiocin is competitive
with ATP and can be overcome at very high levels of ATP.
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DNA Topoisomerases

The mechanism
of DNA gyrase is quite
different from the type
I topoisomerases. A
relaxed molecule is
first folded by the
enzyme to generate a
positive node (a site
where the upper strand
crosses to the left) and
a negative node in
which the upper
strand crosses to the
right. Then, rather
than relieving the
negative turn
induced by a nickingclosing mechanism,
DNA gyrase converts
a positive node into a
negative node by
breaking both strands
of the upper helix,
passing the lower
helix through the
break and then resealing the break. This double-strand passage mechanism converts,
what was a positive twist in the DNA, into a negative twist. This results in the
generation of two negative turns in a single enzymatic event.
Regulation of Superhelical Density of DNA
While DNA gyrase is capable of supercoiling DNA in vitro and novobiocin
and nalidixic acid inhibit first replication and secondly transcription in the cell, the
exact role of gyrase in these three processes is not completely clear.
DNA gyrase could clearly be useful at a replication fork where the parental
DNA strands are being unwound in order to maintain the negative superhelical
density of the DNA ahead of the replication fork. Such an activity could help
facilitate the unwinding of the DNA at the fork itself.
One could also imagine roles for DNA gyrase during both the initiation of
transcription and during the synthesis of RNA as well. However, whether there is
any direct role of the enzyme other than just maintaining the negative superhelical
state of the DNA is not clear at this point.

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DNA Topoisomerases
What controls the two
antagonistic enzymatic
activities that we have talked
about, the DNA
topoisomerase I, that relaxes
specifically negative
superhelical turns, and the
DNA gyrase, that specifically
induces such turns?
Mutant studies
indicate that both enzymes
are required to maintain the
normal degree of
supertwisting of the bacterial
chromosome.
Mutants in DNA
topoisomerase I from Escherichia coli by themselves have been shown to be lethal
mutations. These mutants can survive if they are associated with mutants that
reduce the level of DNA gyrase.
Because the level of reduction of the gyrase can vary, mutants in DNA
topoisomerase I can have superhelical densities of the DNA which is either greater
than the normal superhelical density of DNA from normal cells or it can be in some
cases less than the normal level if the compensating DNA gyrase mutation is very
severe.
Mutants deficient in DNA gyrase are invariably lethal, as are the effects of
antibiotics such as nalidixic acid or novobiocin.
The role of DNA topoisomerase I appears to primarily be to regulate or
modulate the degree of supertwisting of DNA gyrase.
Several genes have been shown to have either increased or decreased
transcriptional activity in mutants with an altered superhelical density.
Two particular genes whose expression is very sensitive to the superhelical
state of the DNA are the gyrA and gyrB genes that encode the subunits of DNA
gyrase. Under conditions where the bacterial chromosome has a reduced
superhelical density, the level of transcription of the DNA gyrase genes is increased
10 to 50 fold. This results in a homeostatic regulation of the superhelical density of
the bacterial chromosome.

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DNA Topoisomerases
Eukaryotic DNA Topoisomerase II
The presence of a type II DNA topoisomerase has been established in many
eukaryotes. Like the bacterial enzymes these enzymes can change the topological
state of DNA using a double-strand passage mechanism and require ATP for their
action. Furthermore, the role of ATP is to allow the enzyme to recycle since
stoichiometric amounts of the enzyme can alter DNA topology.

Unlike the bacterial counterparts the eukaryotic enzymes only relax

supercoiled DNAs, they do NOT supercoil DNA. The eukaryotic enzymes can relax
either positive or negative supercoils using a double-strand passage mechanism.
The enzyme does not appear to wrap DNA around itself when it binds.
The primary reaction of these enzymes appears to allow the passage of DNA
helices through each other.
Cloning and sequence
analysis of both the
prokaryotic and eukaryotic
type II DNA topoisomerases
indicate that they are closely
related evolutionarily.
While the Eukaryotic
enzymes are comprised of a
dimer of subunits 160180,000 molecular weights,
these large subunits have
homology to both the A and
B subunits of DNA gyrase.

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DNA Topoisomerases

The eukaryotic DNA

topoisomerases are also the
targets of many antibioitics as
well as the targets of many drugs
that have been used as anticancer agents. The mechanism of
these drugs is that they inhibit
the DNA topoisomerases in such
a way as to leave the DNA in a
broken or cleaved state during
DNA replication.
The Role of DNA
Topoisomerase II in
Chromosome Segregation
An important biological
role for eukaryotic DNA
topoisomerase II is in the
segregation of chromosomes at
the end of replication. Varshavsky and others have shown that the immediate
products of viral DNA replication are interlocked DNA circles which much be
segregated. Holms and Botstein working in yeast have shown that mutants in DNA
topoisomerase II in yeast are defective in chromosome segregation. Cozzarelli's
group has shown an essential role for DNA topoisomerase IV in segregation of
bacterial chromosomes as well.

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DNA Topoisomerases
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