BMB801 Fall 2020

Lecture Period 4
DNA Topology

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Lecture 4 - Expectations
After lecture 4 (DNA Topology), you should:

1. Understand why the control of DNA topology is

biologically significant.

2. Understand some properties of DNA topology

and the relationship between DNA twist and
supercoiling.

3. Appreciate the influence of DNA topology on

DNA behavior (migration) during electrophoresis.

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Relevance of DNA Topology to Cellular Function
DNA Topology is altered by cellular processes that use DNA
Cellular processes that cause torsional stress on DNA lead to altered
topology. Conversely, changes in DNA topology can alter the rate of many
cellular processes, and may be used as a regulatory mechanism. Some of
these processes include:

1. DNA packaging - genomic DNA compaction by nucleosomes tends to

favor under winding of the DNA helix.

2. DNA replication - the process of replication fork movement creates

underwound and overwound DNA that changes supercoiling ahead and
behind the fork.

3. RNA transcription - the opening of the transcription bubble to generate a

single stranded template leads to supercoiling ahead of the bubble.

4. DNA recombination - DNA strand invasion and extension during

recombination leads to altered DNA topology (Holliday junction) and
changes in supercoiling.

5. Cell Division - newly synthesized DNA generated by replication needs to
be separated for successful completion of cell division.
Figure 1. Supercoiling of cellular DNA is governed by DNA
topoisomerases that act in DNA processes requiring strand
separation (e.g. DNA replication, DNA-dependent RNA
transcription, recombination).
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 DNA Can Adopt Different Topological Forms
DNA can be either relaxed or supercoiled (negative or positive)
DNA in cells is usually negatively supercoiled
Negative supercoiling of DNA can be thought of as a store of free energy
Positive node

that aids in processes that require DNA strand separation. Regions of

(left handed)
DNA that are negatively supercoiled have a tendency to partially unwind.
Consequently, strand separation during replication or transcription can
be accomplished more easily in negatively supercoiled DNA than in
relaxed DNA. Negative node

(right handed)
Interestingly, DNA from some thermophilic microorganisms is positively
supercoiled (helps to prevent DNA denaturation at higher temperatures).

dsDNA
Figure 3. Model of DNA supercoils.
Figure 2. Electron
micrograph image of
relaxed and
supercoiled circular
plasmid DNA.

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Common Mistakes in Supercoil Labeling
Supercoil handedness are often incorrectly labelled

Supplemental Slide
DNA Supercoiling is Induced by Torsional Stress
Supercoiling requires a constraint on the DNA ends
DNA supercoiling occurs in double stranded DNA with
constrained ends
1. DNA constraint can occur by the covalent linkage of DNA
into a circle (circular DNA).
2. Linear DNA can be constrained by their large size that
prevents free rotation of the ends around the central axis.
3. Local regions of DNA can be constrained by binding and
anchorage of DNA to cellular structures (e.g. nuclear
scaffold).

Supercoiling is induced by torsional stress in DNA

containing an intact phosphodiester backbone
1. Physical strand separation, such as during DNA replication
or DNA transcription, induces positive supercoils.
2. Chromatin remodeling, such as during the assembly and
disassembly of nucleosomes and twist during remodeling,
induces both negative and positive supercoils.
3. Enzymatic processes that break the intact phosphodiester
backbones usually relaxing supercoils.
Figure 4. Model for topoisomerase function and topological stress associated with DNA
replication. The replication machinery is represented by a rod moving through the double helix.
DNA ends are anchored to hypothetical immobile structures existing in the nucleus. Upon
initiation of DNA replication, the two strands of duplex DNA are separated, and the replication fork
is formed. Movement of the replication machinery through the immobilized DNA template strands
induces acute overwinding (i.e. positive supercoiling) ahead of the fork. If the replisome rotates
around the helical axis of the DNA, compensatory underwinding (i.e. negative supercoiling)
behind the replication machinery allows some of the torsional stress in the prereplicated DNA to
be translated to the newly replicated daughter molecules in the form of precatenanes. If these
precatenanes are not resolved, they ultimately lead to the formation of catenated duplex daughter
chromosomes. Topoisomerase I is proposed to work ahead of the replication fork to remove
positive DNA supercoils, whereas topoisomerase II is proposed to work primarily behind the fork
to remove precatenanes. In contrast to replication models, drug models place topoisomerase II
ahead of DNA tracking systems (see also http://www.jbc.org/content/280/47/39337/
F1.expansion.html). J.C. Wang (2002) Cellular roles of DNA topoisomerases: a molecular
perspective. Nature Reviews 3: 430-440. 5
Mathematical Consideration of DNA Topology
The Linking Number is Composed of “Twist” and “Writhe”
Note: the physical
In covalently closed circular DNA, the two strands cannot be separated separation of DNA Note: regions of negatively supercoiled DNA tend
from each other without breakage of the phosphodiester backbone. strands in the absence
of topo activity
to partially unwind. This property is useful to
biological processes that depend upon strand
generates positive separation.
supercoils
To separate the strands completely, one strand must pass through the
other (a reaction performed by topoisomerases). The number of times that
one strand would have to pass through the other to separate the strands
is called the “Linking Number”. The linking number (Lk) is composed of
two geometric components called the twist (Tw) and Writhe (Wr).

This property is represented by the equation:

Lk = Tw + Wr

Twist is the number of helical turns that one strand completely wraps
around the other strand. The cross-overs in a right-handed double
stranded helix are defined as positive, such that the linking number of
relaxed DNA (no writhe) will have a positive value
Note: Topoisomerases catalyze breaks in the
Writhe refers to the crossing over of a double stranded helix over itself. phosphodiester backbone to change the linking
number

Right-handed writhe (supercoil) is negative.

Figure 5. Schematic representation of the relationship between DNA
Left-handed writhe is positive. twist and DNA writhe.

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Topoisomerases Change the Linking Number
Changes the linking number requires breaking the DNA backbone
Additional Notes:

1. In the absence of phosphodiester bond breakage,

the linking number for a constrained DNA is
invariant and is equal to the sum of the twist plus
writhe.

2. The only way to change the linking number is to

break the phosphodiester backbone (i.e. simply
changing the twist or writhe will not change linking
number).

3. Topoisomerases change the linking number by

enzymatically breaking the phosphodiester
backbone, rotating the DNA, and resealing the nick.

4. If the ends of the nicked DNA are not free to

rotate around the other intact strand, then the linking Figure 6. Schematic representation of DNA relaxation by
nicking the phosphodiester backbone and swiveling around
number and supercoiling status of the DNA will be a pivot point in the opposite strand.
preserved after strand repair by ligase.

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DNA Topology can be Experimentally Examined
DNA of same length but different linking numbers are “topoisomers”

DNA topoisomers that differ by

a linker number of 1 can be
resolved by agarose gel
relaxed circular

electrophoresis (nicked or covalently closed)

The greater the writhe, the

more compact the DNA. More
compact DNA can migrate linear
more quickly through the
agarose matrix (especially at
partially supercoiled
high voltages).

Figure 8. Ethidium bromide stained plasmid DNA showing migration

differences in agarose gel electrophoresis. (Staining performed after
electrophoresis). Negatively supercoiled pBR322 plasmid DNA was
incubated with reverse gyrase for the indicated times. The extent of
positive supercoiling was monitored by agarose gel electrophoresis.
McClendon et al 2005. J. Biol. Chem. DOI 10.1074/jbc M503320200.

highly supercoiled
Figure 7. Schematic representation of DNA behavior
in agarose gel electrophoresis.
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Breakout Room Session
Physiological implications of topoisomerase activity?
Question 1: What does the data indicate for relative activity
of topoisomerase II on negative supercoils (-SC) versus
positive supercoils (+SC)? What is your conclusion and
what observations support your conclusion?

Question 2: Why does the highly supercoiled form (FI) not

reappear at the longer time points (like the previous
example with reverse gyrase)?

Question 3: What are the potential implications of your

conclusion for topoisomerase II function at DNA
replication forks? Could this enzyme potentially function
ahead of a replication fork to relieve torsional stress?
FIGURE.
Ethidium bromide-stained agarose gels displaying representative time courses
for relaxation of negatively supercoiled ((-)SC; top gel) or positively
supercoiled ((+)SC; bottom gel) pBR322 plasmid DNA by human
topoisomerase IIα in 100 mm KCl are shown. Assay mixtures contained 1 nm
enzyme and 5 nm plasmid. The positions of supercoiled plasmid DNA (form I,
FI) and relaxed DNA (form II, FII) are indicated. Because reactions with both
substrates were processive, DNA relaxation was quantified by the loss of
supercoiled substrate. Error bars represent the standard deviations of four
independent assays.
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DNA Adopt Different Forms of Supercoiling
Interwound (plectonemic) vs. Spiral (solenoid, toroid) Writhe
Plectonemic writhe Toroidal writhe

Writhe can take two forms:

1. Interwound (plectonemic) writhe - the long

axis is twisted around itself.

2. Spiral (solenoid/toroidal) writhe - the long

axis is wound in a cylindrical manner, such as
around a protein.

Figure 9 . A schematic representation of DNA writhe.

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Nucleosome Packaging Introduces Supercoiling
The DNA in a nucleosome is present in a negative supercoil
DNA in a nucleosome is wrapped in a left handed solenoid
= negative supercoil
Plectronic and solenoidal writhe are interconvertible
left handed toroidal coil = right handed plectonemic coil
= negative supercoils

right handed left handed (-) supercoil

Interwound
writhe Spiral
writhe

Figure 10. Crystal structure of the nucleosome showing the left-handed

wrapping of the DNA around the core histones. Figure from http://
www.natureinstitute.org/txt/st/mqual/genome_3.htm Figure 11. Different forms of DNA writhe are interconvertible.

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Nucleosome Packaging Introduces Negative Supercoiling
DNA in a nucleosome is present in a left handed solenoid
Nucleosome assembly is linked to changes in DNA supercoiling.

The wrapping of DNA around the histone core proteins in a

nucleosome (during nucleosome assembly of DNA with constrained
ends) introduces torsional strain

Nucleosome assembly itself does not break the DNA and thus does not
change the linking number.

The torsional strain associated with formation of the negative supercoil

(around the nucleosome) is relieved by a compensatory positive
(plectonemic) supercoil elsewhere.

In eukaryotic organisms, positive supercoils are relaxed by

topoisomerases (decreasing the linking number) while leaving the
negative supercoil associated with the nucleosome unaffected.

This property tends to lead to an overall negative supercoiling of http://www.bioinfo.org.cn/book/biochemistry/chapt23/sim3.htm

Figure 12 (a) Chromatin assembly on relaxed, closed-circular DNA. (b) Binding

genomes that utilize nucleosomes for packaging.
of a histone core to form a nucleosome will induce one negative supercoil; but
in the absence of any strand breaks, a positive supercoil must also form
elsewhere in the DNA (ΔLk = 0). (c) Relaxation of this positive supercoil by
cellular
7 topoisomerases leaves one net negative supercoil (ΔLk = -1).

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