DoubleStranded DNA Can Wrap Around Itself to Form Supercoiled Structures

Figure 27.18. Conserved Residues among Helicases. A comparison of the amino acid sequences of hundreds of
helicases revealed seven regions of strong sequence conservation (shown in color). When mapped onto the
structure of PcrA, these conserved regions lie along the interface between the A1 and B1 domains and along the
ATP binding surface.
III. Synthesizing the Molecules of Life
27. DNA Replication, Recombination, and Repair
27.3. Double-Stranded DNA Can Wrap Around Itself to Form Supercoiled Structures
The separation of the two strands of DNA in replication requires the local unwinding of the double helix. This local
unwinding must lead either to the overwinding of surrounding regions of DNA or to supercoiling. To prevent the strain
induced by overwinding, a specialized set of enzymes is present to introduce supercoils that favor strand separation.
27.3.1. The Linking Number of DNA, a Topological Property, Determines the Degree of
Supercoiling
In 1963, Jerome Vinograd found that circular DNA from polyoma virus separated into two distinct species when it was
centrifuged. In pursuing this puzzle, he discovered an important property of circular DNA not possessed by linear DNA
with free ends. Consider a linear 260-bp DNA duplex in the B-DNA form (Figure 27.19). Because the number of
residues per turn in an unstressed DNA molecule is 10.4, this linear DNA molecule has 25 (260/10.4) turns. The ends of
this helix can be joined to produce a relaxed circular DNA (Figure 27.19B). A different circular DNA can be formed by
unwinding the linear duplex by two turns before joining its ends (Figure 27.19C). What is the structural consequence of
unwinding before ligation? Two limiting conformations are possible: the DNA can either fold into a structure containing
23 turns of B helix and an unwound loop (Figure 27.19D) or adopt a supercoiled structure with 25 turns of B helix and 2
turns of right-handed (termed negative) superhelix (Figure 27.19E).
Supercoiling markedly alters the overall form of DNA. A supercoiled DNA molecule is more compact than a relaxed
DNA molecule of the same length. Hence, supercoiled DNA moves faster than relaxed DNA when analyzed by
centrifugation or electrophoresis. The rapidly sedimenting DNA in Vinograd's experiment was supercoiled, whereas the
slowly sedimenting DNA was relaxed because one of its strands was nicked. Unwinding will cause supercoiling in both
circular DNA molecules and in DNA molecules that are constrained in closed configurations by other means.
27.3.2. Helical Twist and Superhelical Writhe Are Correlated with Each Other
Through the Linking Number
Our understanding of the conformation of DNA is enriched by concepts drawn from topology, a branch of mathematics
dealing with structural properties that are unchanged by deformations such as stretching and bending. A key topological
property of a circular DNA molecule is its linking number (Lk), which is equal to the number of times that a strand of
DNA winds in the right-handed direction around the helix axis when the axis is constrained to lie in a plane. For the
relaxed DNA shown in Figure 27.19B, Lk = 25. For the partly unwound molecule shown in part D and the supercoiled
one shown in part E, Lk = 23 because the linear duplex was unwound two complete turns before closure. Molecules
differing only in linking number are topological isomers (topoisomers) of one another. Topoisomers of DNA can be
interconverted only by cutting one or both DNA strands and then rejoining them.
The unwound DNA and supercoiled DNA shown in Figure 27.19D and E are topologically identical but geometrically
different. They have the same value of Lk but differ in Tw (twist) and Wr (writhe). Although the rigorous definitions of
twist and writhe are complex, twist is a measure of the helical winding of the DNA strands around each other, whereas
writhe is a measure of the coiling of the axis of the double helix, which is called super-coiling. A right-handed coil is
assigned a negative number (negative supercoiling) and a left-handed coil is assigned a positive number (positive
supercoiling). Is there a relation between Tw and Wr? Indeed, there is. Topology tells us that the sum of Tw and Wr is
equal to Lk.
In Figure 27.19, the partly unwound circular DNA has Tw ~ 23 and Wr ~ 0, whereas the supercoiled DNA has Tw ~ 25
and Wr ~ -2. These forms can be interconverted without cleaving the DNA chain because they have the same value of
Lk; namely, 23. The partitioning of Lk (which must be an integer) between Tw and Wr (which need not be integers) is
determined by energetics. The free energy is minimized when about 70% of the change in Lk is expressed in Wr and
30% is expressed in Tw. Hence, the most stable form would be one with Tw = 24.4 and Wr = -1.4. Thus, a lowering of
Lk causes both right-handed (negative) supercoiling of the DNA axis and unwinding of the duplex. Topoisomers
differing by just 1 in Lk, and consequently by 0.7 in Wr, can be readily separated by agarose gel electrophoresis because
their hydrodynamic volumes are quite different supercoiling condenses DNA (Figure 27.20). Most naturally occurring
DNA molecules are negatively supercoiled. What is the basis for this prevalence? As already stated, negative
supercoiling arises from the unwinding or underwinding of the DNA. In essence, negative supercoiling prepares DNA
for processes requiring separation of the DNA strands, such as replication or transcription. Positive supercoiling
condenses DNA as effectively, but it makes strand separation more difficult.
27.3.3. Type I Topoisomerases Relax Supercoiled Structures
The interconversion of topoisomers of DNA is catalyzed by enzymes called topoisomerases which were discovered by
James Wang and Martin Gellert. These enzymes alter the linking number of DNA by catalyzing a three-step process: (1)
the cleavage of one or both strands of DNA, (2) the passage of a segment of DNA through this break, and (3) the
resealing of the DNA break. Type I topoisomerases cleave just one strand of DNA, whereas type II enzymes cleave both
strands. Both type I and type II topoisomerases play important roles in DNA replication and in transcription and
recombination.
Type I topoisomerases catalyze the relaxation of supercoiled DNA, a thermodynamically favorable process. Type II
topoisomerases utilize free energy from ATP hydrolysis to add negative supercoils to DNA. The two types of enzymes
have several common features, including the use of key tyrosine residues to form covalent links to the polynucleotide
backbone that is transiently broken.
The three-dimensional structures of several type I topoisomerases have been determined (Figure 27.21). These structures
reveal many features of the reaction mechanism. Human type I topoisomerase comprises four domains, which are
arranged around a central cavity having a diameter of 20 Å, just the correct size to accommodate a double-stranded DNA
molecule. This cavity also includes a tyrosine residue (Tyr 723), which acts as a nucleophile to cleave the DNA
backbone in the course of catalysis.
From analyses of these structures and the results of other studies, the relaxation of negatively supercoiled DNA
molecules are known to proceed in the following manner (Figure 27.22). First, the DNA molecule binds inside the cavity
of the topoisomerase. The hydroxyl group of tyrosine 723 attacks a phosphate group on one strand of the DNA backbone
to form a phosphodiester linkage between the enzyme and the DNA, cleaving the DNA and releasing a free 5 -hydroxyl
group.
With the backbone of one strand cleaved, the DNA can now rotate around the remaining strand, driven by the release of
the energy stored because of the supercoiling. The rotation of the DNA unwinds supercoils. The enzyme controls the
rotation so that the unwinding is not rapid. The free hydroxyl group of the DNA attacks the phosphotyrosine residue to
reseal the backbone and release tyrosine. The DNA is then free to dissociate from the enzyme. Thus, reversible cleavage
of one strand of the DNA allows controlled rotation to partly relax supercoiled DNA.
27.3.4. Type II Topoisomerases Can Introduce Negative Supercoils Through Coupling
to ATP Hydrolysis
Supercoiling requires an input of energy because a supercoiled molecule, in contrast with its relaxed counterpart, is
torsionally stressed. The introduction of an additional supercoil into a 3000-bp plasmid typically requires about 7 kcal
mol-1.
Supercoiling is catalyzed by type II topoisomerases. These elegant molecular machines couple the binding and
hydrolysis of ATP to the directed passage of one DNA double helix through another that has been temporarily cleaved.
These enzymes have several mechanistic features in common with the type I topoisomerases.
The topoisomerase II from yeast is a heart-shaped dimer with a large central cavity (Figure 27.23). This cavity has gates
at both the top and the bottom that are crucial to topoisomerase action. The reaction begins with the binding of one
double helix (hereafter referred to as the G, for gate, segment) to the enzyme (Figure 27.24). Each strand is positioned
next to a tyrosine residue, one from each monomer, capable of forming a covalent linkage with the DNA backbone. This
complex then loosely binds a second DNA double helix (hereafter referred to as the T, for transported, segment). Each
monomer of the enzyme has a domain that binds ATP; this ATP binding leads to a conformational change that strongly
favors the coming together of the two domains. As these domains come closer together, they trap the bound T segment.
This conformational change also forces the separation and cleavage of the two strands of the G segment. Each strand is
joined to the enzyme by a tyrosine-phosphodiester linkage. Unlike the type I enzymes, the type II topoisomerases hold
the DNA tightly so that it cannot rotate. The T segment then passes through the cleaved G segment and into the large
central cavity. The ligation of the G segment leads to release of the T segment through the gate at the bottom of the
enzyme. The hydrolysis of ATP and the release of ADP and orthophosphate allow the ATP-binding domains to separate,
preparing the enzyme to bind another T segment. The overall process leads to a decrease in the linking number by two.
The degree of supercoiling of DNA is thus determined by the opposing actions of two enzymes. Negative supercoils are
introduced by topoisomerase II and are relaxed by topoisomerase I. The amounts of these enzymes and their activities
are regulated to maintain an appropriate degree of negative supercoiling.
The bacterial topoisomerase II (often called DNA gyrase) is the target of several antibiotics that inhibit the
prokaryotic enzyme much more than the eukaryotic one. Novobiocin blocks the binding of ATP to gyrase.
Nalidixic acid and ciprofloxacin, in contrast, interfere with the breakage and rejoining of DNA chains. These two gyrase
inhibitors are widely used to treat urinary tract and other infections. Camptothecin, an antitumor agent, inhibits human
topoisomerase I by stabilizing the form of the enzyme covalently linked to DNA.
III. Synthesizing the Molecules of Life
27. DNA Replication, Recombination, and Repair
27.3. Double-Stranded DNA Can Wrap Around Itself to Form Supercoiled Structures
Figure 27.19. Linking Number. The relations between the linking number (Lk), twisting number (Tw), and writhing
number (Wr) of a circular DNA molecule revealed schematically. [After W. Saenger, Principles of Nucleic Acid
Structure (Springer-Verlag, 1984), p. 452.]
III. Synthesizing the Molecules of Life
27. DNA Replication, Recombination, and Repair
27.3. Double-Stranded DNA Can Wrap Around Itself to Form Supercoiled Structures
Figure 27.20. Topoisomers. An electron micrograph showing negatively supercoiled and relaxed DNA. [Courtesy of
Dr. Jack Griffith.]
III. Synthesizing the Molecules of Life
27. DNA Replication, Recombination, and Repair
27.3. Double-Stranded DNA Can Wrap Around Itself to Form Supercoiled Structures
Figure 27.21. Structure of a Topoisomerase. The structure of a complex between a fragment of human topoisomerase I
and DNA.
III. Synthesizing the Molecules of Life
27. DNA Replication, Recombination, and Repair
27.3. Double-Stranded DNA Can Wrap Around Itself to Form Supercoiled Structures
Figure 27.22. Topoisomerase I Mechanism. On binding to DNA, topoisomerase I cleaves one strand of the DNA
through a tyrosine (Y) residue attacking a phosphate. When the strand has been cleaved, it rotates in a controlled manner
around the other strand. The reaction is completed by religation of the cleaved strand. This process results in partial or
complete relaxation of a supercoiled plasmid.
III. Synthesizing the Molecules of Life
27. DNA Replication, Recombination, and Repair
27.3. Double-Stranded DNA Can Wrap Around Itself to Form Supercoiled Structures
Figure 27.23. Structure of Topoisomerase II. A composite structure of topoisomerase II formed from the aminoterminal ATP-binding domain of E. coli topoisomerase II (green) and the carboxyl-terminal fragment from yeast
topoisomerase II (yellow). Both units form dimeric structures as shown.
III. Synthesizing the Molecules of Life
27. DNA Replication, Recombination, and Repair
27.3. Double-Stranded DNA Can Wrap Around Itself to Form Supercoiled Structures