DNA Linking number

DNA TOPOLOGY
• The double-stranded structure of a DNA determines its biological
function.
• Metabolic events involving unwinding impose great stress on the DNA
because of the constraints inherent in the double helix.
• Molecular processes such as replication and transcription require the
unwinding of the DNA double helix.
• While unwinding, correct topological tension in the DNA (super-helical
density) should be maintained in order for genes to be regulated and
expressed normally.
• In 1965, Vinograd et al. discovered that the circular DNA chromosomes
isolated from small viruses such as SV40 or polyoma virus were in a
highly compact or folded conformation.
• This supercoiling or writhing of the circular DNAs was a result of the
DNAs being under wound with respect to the relaxed form of DNA.
• When a linear DNA is free in solution, it assumes a pitch that contains
10.4 base pairs per turn.
• As the result, the DNA is less tightly wound than 10.0 base pairs per turn
in the Watson and Crick B-form DNA.
• In order to understand the origin of supercoiling; imagine a linear DNA of
4,200 base pairs in length.
• If the DNA were in the B-form, one would expect the two strands of the
helix to be wrapped around each other 400 times (4,200 bp/10.4 bp/ turn).
• Imagine a linear DNA in which the two ends become connected to form an
open circle.
• This is referred to as a relaxed circular DNA.
• On the other hand, if the linear DNA were unwound 10 per cent, say 40
turns, before its ends were joined, then the DNA molecule would be under
stress.
• When the molecule is free in solution, it will coil about itself in space, as the
two strands simultaneously twist about each other in order to return to
equilibrium value of 10.4 base pairs per turn.
• The DNA that is ‘underwound is
referred to as negatively
supercoiled’.

• The DNA in this case forms the

right-handed double helices.

• The DNA that is ‘overwound is

referred to as a positively
supercoiled DNA helix and is a
left-handed helix’ (Figure 1.5).
Linking number
• The total number of times one strand of the DNA helix is linked with the other in
a covalently closed circular molecule is known as the ‘linking number (Lk)’
(Figure 1.6).
Salient features about linking number
• The Lk is only defined for a covalently closed DNA and its value is fixed
provided the molecule remains covalently closed.
• The Lk does not change whether the covalently closed circle is forced to
lie in a plane in a stressed conformation or it is allowed to supercoil about
itself freely in space.
• The Lk of a circular DNA can only be changed by breaking a
phosphodiester bond in one of the two strands, allowing the intact strand
to pass through the broken strand and then rejoining the broken strand.
• Lk is always an integer since two strands must always be wound about
each other an integral number of times upon closure.
•
Twists and writhes
• ‘The number of times the two strands of DNA are twisted about each
other is called the twists (Tws).
• The writhes (Wrs) are the number of times that the DNA helix is coiled
about itself in three-dimensional space.’
• Tw and Wr are geometric rather than topological properties.
• The Tw and the Wr are not necessarily integers. It is just their sum, the Lk
that is an integer.

Lk = Tw + Wr
 • The DNA length and its pitch in solution determine the Tw of DNA.
Tw = length (bp)/pitch (bp/turn).
• The Tw and the Lk determine the value of the Wr.
` Wr = Lk - Tw

• Unlike the Tw and the Lk, the Wr of a DNA only depends on the path the
helix axis takes in space.
• If the path of the DNA is in a plane, the Wr is always zero.
• In addition, if the path of the DNA helix were on the surface of a sphere,
then the total Wr can also be shown to be zero.
• Wrs can come in different forms.

• If a DNA molecule wrap around itself, then

the Wrs are known as supertwists.

• If a DNA molecule wrap around something

else (another molecule for instance), then
the Wrs are known as ‘solenoidial’ Wrs.

• In solution, the Wrs can isomerize between

the supertwist and solenoidal forms.
Measuring supercoils
•
• A s of 0.1 means that 10 per cent of the helical turns in a sample of DNA (in
its B configuration) have been removed.
• This underwinding results in negative supercoiling. In a cell, σ is usually of
5–7 per cent.
• The superhelical density of a circular DNA can be observed and measured
in several ways; for example, electron microscopy, sedimentation velocity
or electrophoresis.
• Supercoiling can be measured by sedimentation procedure.
• Since supercoiled molecules are more compact, they sediment faster in a
centrifuge than when they are relaxed.
• Supercoiling can also be determined by electrophoresis in an agarose gel.
• A supercoiled DNA migrates much more rapidly than does a relaxed
molecule of the same length.
• The DNA separates into discrete bands depending on the Lk.
• Since the DNAs resolved in this way differ from each other only in their
topology, they are referred to as ‘topological isomers or topoisomers’.
• Molecules that differ by one unit in Lk can be separated by electrophoresis
in agarose due to the difference in their Wr (that is due to difference in
folding).
Topoisomerase.
 Topoisomerases
• ‘Topoisomerases’ are enzymes that change the Lk of a circularly wound
double-stranded DNA.
• The change in Lk changes the Wr.
• The variation in Wr subsequently changes the state of the compaction of the
DNA molecule.
• The naturally occurring DNA is underwound or negatively supercoiled.
• This is advantageous because it permits the DNA to be transiently and locally
melted to permit the enzymes of the DNA replication and transcription to
copy and synthesize new DNA or RNA.
• There are two classes of topoisomerase: Type I and Type II.
Type I topoisomerases
• These enzymes remove supercoils by breaking only one of the two
strands of the DNA.
• As a result, these enzymes change the Lk by 1 each time.
• The best-characterized member of this class in E. coli is Topoisomerase I.
• This enzyme is 864 amino acids in length and is monomeric; it is encoded
by the topA gene.
• The mechanism of catalysis involves the formation of a covalent
intermediate between a tyrosine residue and the phosphodiester
backbone.
• Specifically, nucleophilic attack from the hydroxyl group of tyrosine to a phosphorus
atom creates a phosphodiester link between the enzyme and the DNA and generates a
free 5ʹ-hydroxyl group.
• Formation of this bond is energetically neutral as the reaction involves the
replacement of one phosphomonoester bond with another—such reactions are called
‘transesterification’ reactions.
• The other strand of the DNA is held in place by binding non-covalently to a domain of
the enzyme.
• The cleaved strand is resealed.
• Thus, one supercoil is removed.
• Topo I from E. coli acts only on negative supercoils; while eukaryotic Topo I can remove
both negative and positive supercoils (Figure 1.7).
Topoisomerase I
Type II topoisomerases
• These enzymes act through a mechanism, in which both the phosphodiester
backbone chains are broken simultaneously.
• As a result, the Lk changes by two.
• Some Type 2 enzymes can use ATP to introduce the superhelical turns into the
DNA.
• The best-characterized member of this class is E. coli Topoisomerase II—better
known as DNA gyrase.
• E. coli DNA gyrase is a tetrameric protein consisting of two A subunits (875 aas)
and two B subunits (804 aas).
• Depending on the DNA substrates, these enzymes can change positive supercoils
into negative supercoils or increase the number of negative supercoils by 2.
• Type II topoisomerases catalyse catenation
and decatenation, i.e., linking and unlinking
of two different DNA duplexes.

• The enzyme also introduces negative

supercoils at or near the Ori C site in the
DNA template.

• DNA gyrase also removes the positive

supercoils that are formed ahead of the
growing fork during replication
• DNA gyrase is composed of two identical
subunits.
• The hydrolysis of ATP by gyrase’s inherent
ATPase activity powers the conformational
changes that are critical to the enzyme’s
operation.
• The enzyme is a dimer, which has two
identical subunits.
• Initially, the enzyme binds one part of a DNA
strand, the G segment, inducing a
conformational change in the B, Bʹ, A and Aʹ
domains of the enzyme.
• After the binding of ATP (indicated by the asterisks)
and another part of the DNA strand, the T segment, a
series of reactions occur in which the G segment is cut
by the A and Aʹ domains of the enzyme and the ends
of the G-DNA become covalently linked to tyrosine
residues in these domains.
• Simultaneously, the ATP-binding domains move
towards each other, transporting the T segment
through the break and into the central hole.
• The cut G segment is then
resealed and the T segment is
released by a conformational
change that separates the A and
Aʹ domains at the bottom of the
enzyme.
• The interface between the A and Aʹ 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 the 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.
• Topoisomerases are essential enzymes.
• The mutations of any of the genes coding for topoisomerases are usually
lethal.
• They are, therefore, the targets for the antibiotics and other drugs.
• Bacteria can be killed by novobiocin or nalidix acid.
• Both of these inhibit DNA gyrase.
• Novobiocin blocks ATP binding and nalidixic acid blocks the breakage and
rejoining mechanism.
• These antibiotics do not inhibit eukaryotic topoisomerases and can be used
to eradicate bacterial infections.
• Some bacteria, however, are now resistant to novobiocin.
• Eukaryotic topoisomerase inhibitors, such as doxorubicin and etoposide, are
used as chemotherapeutic agents.