DNA Topology and Supercoiling

DNA Topology and Supercoiling
1
 Topology is a mathematical science that studies the possible
conformation that DNA can assume in the 3D space. Keep in mind
that DNA is an ordered while still flexible structure. Its exact
molecular parameters are a function of both the surrounding ionic
environment and the nature of the DNA-binding proteins with which
it is complexed.
 Understanding the topology of DNA and how the cell both

accommodates and exploits topological constraints during DNA
replication, transcription, and other chromosomal transactions is of
fundamental importance in molecular biology.
2
Problem:
Measured linearly, the Escherichia coli genome (4.6 Mb) would be 1,000
times longer than the E. coli cell.
The human genome (3.4 Gb) would be 2.3 m long if stretched linearly.
Figure 4.1
Chromosome released
from lysed E. coli cell.
Solution: Dna contained in

procaryotic and eucaryotic cells
is tightly compacted through
various supercoiling degree.
3
Relaxed vs. Supercoiled DNA
Relaxed DNA
The picture on the left shows the unique

E. Coli chromosome in a relaxed state
and at increasing degree of supercoiling.
Supercoiled DNA
4
Supercoiling of DNA Shown by Gel
Electrophoresis
Agorose Gel Stained with Ethidium Bromide
Supercoiled circular DNA migrates faster Ethidium = fluorescent

intercalating agent used to stain
during agarose gel electrophoresis than does DNA in agarose gels
relaxed circular DNA (or linear DNA).
5
6
Formation of DNA Supercoil
Covalently closed, circular DNA
(cccDNA) is topologically
constrained, because the ends of
the two strand are not free to
rotate to accommodate changes
in the number of times the two
chains of the double helix twist
about each other. Topologically
constrained structures, such as
cccDNA, are characterized by a
topological property called
Linking Number.
7
Linking Number
L=T+W
• L or Lk = linking number (number of times one strand crosses the other). The
number of times one strand would have to be passed through the other strand
in order for the two strands to be entirely separated from each other is called
the linking number. It is a fixed topological property for topological constrained
DNA.
• T = twist (number of helical turns; for B-DNA, T = # bp divided by ~10.5 bp/turn)
• W = writhe (number of supercoils)
L0 = linking number of relaxed molecule = T, since W = 0 in relaxed

molecule
L=linking difference = L-L0
Superhelical Density: s = L/L0 = W/T; it is a normalized measure 8
Strand separation of a topologically contrained DNA (as is the case of linear
chromosomes), causes torsional accumulation stress that results in supercoiling. This is
what happens when DNA is unzipped to allow replication and transcription. A molecule
of cccDNA can readily undergo distortions that convert some of its twist to writhe or
some of its writhe to twist without the breakage of any covalent bonds.
9
Type I Topoisomerases
•∆L = ±1 per cycle

•Cleaves a single strand
•Passes broken single
strand around the other,
then rejoins strands
•Does not require ATP
Ο Ο •Relaxes supercoiled
Ο Ο
DNA
10
Structure of a Type I Topoisomerase
11
Type II Topoisomerases
•∆L = ±2 per cycle

•Cleaves both strands
•Passes unbroken part of
duplex through double- strand
break, then rejoins strands
•Requires ATP
•Relaxes supercoiled DNA
•Some type II enzymes (like
DNA gyrase) can add negative
supercoils
12
Type II topoisomerase make transient double-strand breaks in the DNA through
which they pass a segment of uncut duplex DNA before resealing the break.
13
X-Ray Crystal Structure of a Type II
Topoisomerase
14
Topological Interconversions Catalyzed by
Type II Topoisomerase
Relaxation
Catenation and
Decatenation
Knotting and
Unknotting
15
Negative Supercoiling of DNA is Important
Biologically
• Negatively supercoiled DNA is equivalent to underwound DNA. This

makes it easier to separate strands during replication and
transcription.
• In eukaryotes, formation of nucleosomes results in torsional strain in the

DNA molecule (equivalent to ~1.5-1.8 supercoils/nucleosome particle
theoretically; actual value is ~1), which is relieved by topoisomerases. This
results in DNA that is negatively supercoiled once histone proteins are
removed.
• In prokaryotes, an enzyme called DNA gyrase (a type II

topoisomerase) generates negative supercoils.
16
Chromosomes and Chromatin: Higher-
Order DNA Structure
17
Eukaryotic chromosome structure
Chromatin complex of DNA and chomosomal proteins

~ twice as much protein as DNA
Two major types of proteins:
1. Histones abundant, basic proteins with a positive charge

that bind to DNA
5 main types: H1, H2A, H2B, H3, H4
~equal in mass to DNA
evolutionarily conserved
2. Non-histones all the other proteins associated with DNA
differ markedly in type and structure
amounts vary widely

>> 100% DNA mass
<< 50% DNA mass
18
19
Metaphase chromosome depleted of histones maintains its shape with a
nonhistone protein scaffold.
20
Nucleosome
 The basic repeating unit of

chromatin.
 It is made up by five histone
H3 proteins: H2A, H2B, H3, H4 as
H2A H2B core histones and H1 as a linker.
H4  It provides the lowest level of
compaction of double-strand DNA
into the cell nucleus.
 It often associates with
transcription.
1974: Roger Kornberg discovers nucleosome who won Nobel Prize in 2006.
21
22
Core Histones are highly conserved proteins - share a structural motif called a
histone fold including three α helices connected by two loops and an N-terminal tail
23
Nucleosome Assembly In Vitro
4 core histones + 1 naked DNA template at 4C at 2M salt concentration,

from Dyer et al, Methods in Enzymology (2004), 375:23-44.
24
25
The N-terminal tails protrude from the core
26
Histone Modifications
Epigenetic Modifications
Acetylation
Me
Ac
Me Methylation
Ub
‘Histone Code’
Ubiquitination
Su
Sumoylation
P
Phosphorylation
27
Histone post-translational modification regulates
chromatin relaxation/compactation
Histone acetylation
alters the conformation
of chromatin structure
in nucleus by relaxing
the chromatin and
allowing transcriptional
activation. It is
regulated by two sets
of enzymes HATs and
HDACs which add or
remove acetyl group
respectively from both
histone and non-
histone proteins, hence
regulating gene
transcription.
28

DNA Topology and Supercoiling

Uploaded by

Copyright:

Available Formats

DNA Topology and Supercoiling

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

DNA Topology and Supercoiling

Uploaded by

Copyright:

Available Formats

DNA Topology and Supercoiling

 Understanding the topology of DNA and how the cell both

Solution: Dna contained in

The picture on the left shows the unique

Supercoiled circular DNA migrates faster Ethidium = fluorescent

L0 = linking number of relaxed molecule = T, since W = 0 in relaxed

•∆L = ±1 per cycle

•∆L = ±2 per cycle

• Negatively supercoiled DNA is equivalent to underwound DNA. This

• In eukaryotes, formation of nucleosomes results in torsional strain in the

• In prokaryotes, an enzyme called DNA gyrase (a type II

Chromatin complex of DNA and chomosomal proteins

Two major types of proteins:

1. Histones abundant, basic proteins with a positive charge

5 main types: H1, H2A, H2B, H3, H4

~equal in mass to DNA

2. Non-histones all the other proteins associated with DNA

differ markedly in type and structure

amounts vary widely

 The basic repeating unit of

4 core histones + 1 naked DNA template at 4C at 2M salt concentration,

You might also like