DNA molecules can coil and bend in space, leading to changes in topology, including
formation of negative or positive supercoils. For example, as discussed in Chapter 4, local unwinding of a DNA
duplex whose ends are fixed causes stress that is relieved by supercoiling. The
enzymes that control the topology of DNA function at several different steps in
replication in both prokaryotic and eukaryotic cells. In this section we describe
the two different classes of topoisomerases and their role in DNA replication.
Type I Topoisomerases Relax DNA by Nicking and Then Closing One Strand of
Duplex DNA
Figure 12-14
.
Action of E. coli type I topoisomerase (Topo
I)
The DNA-enzyme intermediate contains a covalent bond between the
5′-phosphoryl end of the nicked DNA and a tyrosine residue
in the protein (inset). After the free 3′-hydroxyl end of
the red cut strand passes under the uncut strand, it attacks the
DNA-enzyme phosphoester bond, rejoining the DNA strand. During each
round of nicking and resealing catalyzed by E. coli
Topo I, one negative supercoil is removed. (The assignment of sign
to supercoils is by convention with the helix stood on its end; in a
negative supercoil the “front” strand falls from
right to left as it passes over the back strand (as here); in a
positive supercoil, the front strand falls from left to right.)
The first
topoisomerase to be discovered,
E. coli topoisom-erase
I, can remove negative supercoils without leaving nicks in the DNA molecule
(). After the
enzyme
binds to a DNA molecule, it cuts one strand, simultaneously generating a
covalent phosphoester bond between the released 5′ phosphate on the
DNA and a tyrosine residue in the
enzyme. Formation of this phosphotyrosine bond
does not require ATP or another source of energy. The free 3′-hydroxyl
end of the DNA is held noncovalently by the
enzyme. The DNA strand that has not
been cleaved is then passed through the single-stranded break. The cleaved
strand is then resealed, forming a structure with the same
chemical bonds as the starting DNA, but with one less
negative supercoil. By this mechanism, the
enzyme removes one negative supercoil
at a time.
Figure 12-15
.
Separation of SV40 DNA topoisomers containing different numbers
of supercoils by gel electrophoresis
DNA was extracted from SV40 virions under conditions that ensure the
maximal number of supercoils (lane 1). Aliquots were treated with
E. coli type I topoisomerase for 3 min (lane 2)
or 30 min (lane 3). About 25 bands, equal to the number of possible
topoisomers, are visible in the electrophoretograms after Topo I
treatment, including the fully relaxed form, which has no
supercoils. Because DNAs with few supercoils tend to assume an
extended, rodlike conformation, they migrate more slowly in gels
than do the more compact molecules that have extensive supercoils.
[From W. Keller and I. Wendel, 1974, Cold Spring Harbor
Symp. Quant. Biol.
39:199; courtesy of W. Keller.]
Any
enzyme that cleaves only one strand of a DNA duplex and then reseals it is
classified as a
type I topoisomerase (Topo I). The Topo I from
E. coli acts on negative, but not positive, supercoils. In
contrast, Topo I
enzymes from eukaryotic cells can remove both positive and
negative supercoils. Because the relaxation (removal) of DNA supercoils by Topo
I is energetically favorable, the reaction proceeds without an energy
requirement. The sequential action of
E. coli Topo I can remove
essentially all the supercoils in a DNA molecule ().
Studies with gene-targeted knockout strains of E. coli have
established that Topo I is essential for viability. The enzyme is thought to
help maintain the proper superhelical density of the E. coli
chromosome by removing negative supercoils formed by action of type II
topoisomerase (DNA gyrase), which is discussed below. Since E.
coli Topo I cannot remove positive supercoils, it is unlikely to
play a role in growing fork progression, which generates positive supercoils. In
yeast, either Topo I or Topo II, both of which can relax positive and negative
supercoils, functions in the movement of growing forks. Mutations in the gene
encoding Topo I affect the growth rate of yeast cells but are not lethal; thus,
this enzyme is not essential for viability. However, yeast Topo II, which
separates the two DNA duplexes following replication, is essential for
viability. In both yeast and E. coli, Topo I enzymes may be
important in regulating aspects of transcription, as well as in repairing
damaged DNA.
Type II Topoisomerases Change DNA Topology by Breaking and Rejoining
Double-Stranded DNA
Figure 12-16
.
Action of E. coli DNA gyrase, a type II
topoisomerase
(a) Introduction of negative supercoils. The initial folding
introduces no stable change, but the subsequent activity of gyrase
produces a stable structure with two negative supercoils. Eukaryotic
Topo II enzymes cannot introduce supercoils but can remove negative
supercoils from DNA. (b) Catenation and decatenation of two
different DNA duplexes. Both prokaryotic and eukaryotic Topo II
enzymes can catalyze this reaction. [See N. R. Cozzarelli, 1980,
Science
207:953.]
The first
type II topoisomerase (Topo II) to be described was
isolated from
E. coli. and named
DNA gyrase.
Topo II
enzymes have the ability to cut both strands of a double-stranded DNA
molecule, pass another portion of the duplex through the cut, and reseal the cut
in a process that utilizes ATP (). Depending on the DNA
substrate, these maneuvers will have
the effect of changing a positive supercoil into a negative supercoil or of
increasing the number of negative supercoils by 2. The Topo II
enzymes from
mammalian cells cannot, like
E. coli DNA gyrase, increase the
superhelical density at the expense of ATP; presumably no such activity is
required in
eukaryotes, since binding of
histones increases the potential
superhelicity. All type II
topoisomerases catalyze
catenation
and
decatenation, that is, the linking and unlinking, of two
different DNA duplexes ().
Figure 12-17
.
Molecular model for the catalytic activity of E.
coli topoisomerase II (DNA gyrase)
The enzyme is a dimer of two identical subunits. Initially, the
enzyme binds one part of a DNA strand, the G segment (dark blue),
inducing a conformational change in the B, B′, A, and
A′ domains of the enzyme 2. After binding of
ATP (indicated by the asterisks) and another part of the DNA strand,
the T segment (light blue), a series of reactions occur in which the
G segment is cut by the A and A′ domains (light orange) of
the enzyme and the ends of the G DNA become covalently linked to
tyrosine residues in these domains 3 and
3a. Simultaneously, the ATP-binding domains (green)
move toward each other, transporting the T segment through the break
and into the central hole 4. The cut G segment is then
resealed, and the T segment is released by a conformational change
that separates the A and A′ domains at the bottom of the
enzyme 5. The interface between the A and A′
domains then re-forms, a reaction that requires ATP hydrolysis and
regenerates the starting state 2. At this point, the G
segment can dissociate from the enzyme by conversion of
2 into 1. Alternatively, the enzyme
can proceed through another cycle, again passing the T segment
through the G segment and thus removing two more supercoils. [From
J. Berger et al., 1996, Nature
379:225.]
Figure 12-18
.
Movement of the growing fork during DNA replication induces
formation of positive supercoils in the duplex DNA ahead of the
fork
In order for extensive DNA synthesis to proceed, the positive
supercoils must be removed (relaxed). This can be accomplished by
E. coli DNA gyrase and by eukaryotic type I and
type II topoisomerases. [Adapted from A. Kornberg and T. Baker,
1992, DNA Replication, 2d ed., W. H. Freeman and
Company, p. 380.]
DNA gyrase is composed of two identical subunits.
Hydrolysis of ATP by
gyrase’s inherent
ATPase activity powers the conformational changes
that are critical to the
enzyme’s operation (). The
enzyme functions to introduce negative
supercoils at or near the
oriC site in the DNA
template; as
noted earlier, DnaA can initiate replication only on a negatively supercoiled
template. Measurements of the degree of DNA supercoiling in
E.
coli suggest that there is one negative supercoil for each
15 – 20 turns of the DNA helix. A second crucial
function of gyrase is to remove the positive supercoils that form ahead of the
growing fork during elongation of the growing strands ().
Replicated Circular DNA Molecules Are Separated by Type II
Topoisomerases
Figure 12-19
.
Completion of replication of circular DNA molecules
Denaturation of the unreplicated terminus followed by supercoiling
overcomes the steric and topological constraints of copying the
terminus. At least with SV40 DNA, the final two steps (synthesis and
decatenation) can occur in either order depending on experimental
conditions. Parental strands are in dark colors; daughter strands in
light colors. (Inset) Electron micrograph of two
fully replicated SV40 DNA molecules interlocked twice. This
structure would result if synthesis was completed before
decatenation. Topo II can catalyze decatenation of such interlocked
circles in vitro. [Drawing adapted from S. Wasserman and N.
Cozzarelli, 1986, Science
232:951. Micrograph from O. Sundin and A. Varshavsky,
1981, Cell
25:659; courtesy of A. Varshavsky.]
During DNA replication the parental strands remain intact and retain their
superhelicity. This poses steric and topological constraints to the completion
of replication of a circular DNA molecule as the two
growing forks approach each
other. (A similar situation arises wherever two
growing forks in a linear
eukaryotic
chromosome approach each other.) illustrates how the last few helical turns in the
parental DNA could be removed by changing the topology of the already replicated
regions, leaving the two nearly complete daughter helices linked together as
catenanes, covalently linked but not yet completely
finished circles. Replication then could be completed before or after
decatenation to yield two separated complete daughter helices.
In
E. coli, decatenation is catalyzed by DNA gyrase and a second
type II
enzyme, called
topoisomerase IV, which genetic studies
suggest is responsible for separating newly replicated molecules
in vivo.
Furthermore, temperature-sensitive Topo IV mutants carrying
plasmids accumulate
interlocked
plasmids — that is, catenanes of
plasmid DNA, which appear similar to the SV40 catenane shown in
(inset). To separate DNA catenanes, Topo IV presumably binds to
the interlocked duplexes and makes a double-stranded break in one molecule;
while remaining attached to the
substrate, it then passes the other molecule
through the break and finally reseals the break in the cut molecule (see Figure
12-17). Interestingly, although DNA gyrase can carry out decatenation
in vitro,
it cannot fully substitute for Topo IV
in vivo, as demonstrated by the lethal
effects of Topo IV
mutations.
Linear Daughter Chromatids Also Are Separated by Type II
Topoisomerases
The use of temperature-sensitive mutations in Topo II has shown the importance of
this protein in yeast-cell viability; furthermore, light-microscopic studies
have revealed the participation of Topo II in separating the linear chromosomes
of yeast cells. Although individual yeast chromosomes are too small to be
visualized by light microscopy, a structure called the nuclear
body, which contains all the chromosomes clumped together, can be
seen. When temperature-sensitive Topo II mutants are shifted to a nonpermissive
temperature, the nuclear body, which usually divides at the junction of the
mother and daughter cell, appears to get stuck in the passageway between the two
cells.
Fluorescent antibody staining of metaphase chromosomes reveals that topoisomerase
II is a principal component of the nonhistone protein scaffolding to which long
DNA loops are attached. This finding and the genetic studies in yeast strongly
suggest that eukaryotic Topo II resolves tangles that exist in newly replicated
linear chromosomes. And finally, the similarity between the phenotypes of Topo
II mutants in yeast and Topo IV mutants in bacteria argues that control of
topological domains may be analogous in eukaryotic chromosomes and in small
circular DNA molecules.
SUMMARY
-
Among the proteins involved in DNA
replication are several that change the topology of DNA: helicases,
which can unwind the DNA duplex, thereby inducing formation of
supercoils, and topoisomerases, which catalyze addition or removal of
supercoils.
-
Type I topoisomerases relax DNA (i.e.,
remove supercoils) by nicking and closing one strand of duplex DNA (see
). -
Type II topoisomerases change DNA topology
by breaking and rejoining double-stranded DNA. These enzymes can
introduce or remove supercoils and can separate two DNA duplexes that
are intertwined (see ). -
Topoisomerases are important both in
growing fork movement and in resolving (untangling) finished chromosomes
after DNA duplication. Both replicated circular and linear DNA
chromosomes are separated by type II topoisomerases.
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