DNA topoisomerases in vivo

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DNA topoisomerasesin vivo

• Dr. Sevim Isik

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What is Supercoiling?
In addition to the helical coiling of the strands
to form a double helix, the double stranded DNA
molecule can also twist upon itself.
Supercoiling occurs in nearly all chromosomes
(circular or linear)
Relaxed DNA has no supercoils 10.4 bp
Negatively supercoiled DNA is underwound (favors
unwinding of the helix) DNA isolated from cells
is always negatively supercoiled
Positively supercoiled DNA is overwound
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The Linking Number (L) of DNA
The linking number of DNA, a topological
property, determines the degree of
supercoiling The linking number defines
the number of times a strand of DNA winds in the
right-handed direction around the helix axis when
the axis is constrained to lie in a plane
If both strands are covalently intact, the
linking number cannot change Only
topoisomerases can change the linking number.
540 bp
100 bp
L54010 54
L(540-100)10 44
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Type I Topoisomerases
They relax DNA by nicking then closing one strand
of dublex. They cut one strand of the double
helix, pass the other strand through, then rejoin
the cut ends.
L n
L n1
Topo I of eukaryotes 1) acts to relax positive
or negative supercoils 2) changes linking number
by 1 or 1 increments
Topo I of E. coli 1) acts to relax only negative
supercoils 2) increases linking number by 1
increments
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Type I mechanism
All topoisomerases cleave DNA using a covalent
Tyrosine-DNA intermediate
Because the relaxation (removal) of DNA
supercoils by Topo I is energetically favorable,
the reaction proceeds without an energy
requirement.
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Type II Topoisomerases
They relax or underwind DNA by cutting both
strands then sealing them. They change the
linking number by increments of 2 or -2
relaxed DNA
DNA Gyrase
Relaxation
Negative Supercoiling
Topo II
(-) supercoiled DNA
Topo II of E. coli (DNA Gyrase) 1) Introduce
negative supercoils or relaxes pos. supercoils 2)
Increases the linking number by increments of
2 3) Requires ATP
Topo II of Eukaryotes 1) Relaxes only negatively
supercoiled DNA 2) Increases the linking number
by increments of 2 3) Requires ATP
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Type II mechanism
Cleavable Complex
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Reactions catalysed by topoisomerases

• Prokaryotes

relaxation
relaxation
supercoiling
Topo II of E. coli (DNA Gyrase) 1) Introduce
neg. supercoils or relaxes pos. supercoils 2)
Increases the of neg. supercoils by increments
of 2 3) Requires ATP
Topo I of E. coli 1) acts to relax only negative
supercoils 2) increases linking number by 1
increments
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Reactions catalysed by topoisomerases

• Eukaryotes

relaxation
relaxation
Topo II of Eukaryotes 1) Relaxes only negatively
supercoiled DNA 2) Increases the supercoiling by
increments of 2 3) Requires ATP
Topo I of eukaryotes 1) acts to relax positive
or negative supercoils 2) changes linking number
by 1 or 1 increments
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Reactions catalysed by topoisomerases
Knotting irreducible entanglement of a single
DNA molecule
Type I or Type II topo
knotting
unknotting
Catenation the linking of two or more DNA
molecules in which at least one strand of each
dublex is in the form of a closed ring
Type II topo
catenation
decatenation
If one strand is nicked, only then topo I
catalyse catanation or decatanation
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Functions of Topoisomerases

• DNA Replication
• Chromatin Condensation
• Segregation of Chromosomes during mitosis and
  meiosis
• Transcription
• Recombination
• DNA Repair

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The role of topoisomerases in replication

• Initiation
• Requirement for supercoiling
• DnaA requires negative supercoiling to work
• Elongation
• Requirement for relaxation of supercoiling in
  front of replicatipon fork
• Requirement for relaxation of excess (-)
  supercoiling behind replication fork
• Termination
• Removal of Catenanes (and precatenanes)

Free rotation can not occur
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Types of topoisomerases in replication

• Prokaryotes
• Initiation
• Gyrase introduce negative supercoils at or near
  the oriC site in the DNA template
• Elongation
• Gyrase relax () supercoiling to introduce (-)
  sc
• Termination
• Gyrase
• Topo IV (a type II topo)

remove catenanes
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Types of topoisomerases in replication

• Eukaryotes
• Initiation
• Gyrase introduce negative supercoils at or near
  the oriC site in the DNA template
• Elongation
• Topo I relax () supercoiling
• Termination
• Topo II? remove catenanes

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Elongation of replication
Precatenanes and () supercoils are formed in
front of replication fork.
leading strand
lagging strand
positive supercoils

precatenanes

negative supercoils
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Elongation of replication
Eukaryotes Topo I relaxes positive supercoils
ahead of replication fork
Topo I
Relaxation of () sc by topo I
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Elongation of replication
Prokaryotes DNA Gyrase remove positive
supercoils that normally form ahead of the
growing replication fork by adding negative
supercoils
DNA gyrase
E. Coli ? DNA gyrase (adds neg. supercoils)
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Termination of replication
Topo II removes precatenanes at the end of
replication
Type
precatenanes
Prokaryotes topo IV Eukaryotes topo II?

Type II topoisomerases
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The role of topoisomerases in recombination
After DNA duplication, the chromosome pairs line
up in a tetrad configuration .Adjacent
chromosomes can exchange parts. Exchanging parts,
simply mean that they exchange stretches of DNA.
DNA replication and recombination generate
intertwined DNA intermediates that must be
decatenated for chromosome segregation to occur.
Bacteria Topoisomerase IV (topo IV) is the
decatenase of DNA recombination intermediates.
The function of topo IV is dependent on the
level of DNA supercoiling. The role of gyrase in
decatenation is to introduce negative supercoils
into DNA, which makes better substrates for topo
IV. Eukaryotes Topo II? decatenates the
intertwined DNA intermediates. Topo I relaxes
overwound DNA.
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Chromosome Segregation (decatanation)
Replicated DNA molecules are separated
(decatenated) by type II topoisomerases
Catenated (linked)
topo IV
E. Coli topo IV , Eukaryotes topo II??
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Condensation cycle during replication
Decondensation
Replication
Chromosome segregation
Condensation
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The role of topoisomerases in condensation

• Bacteria
• free (-) supercoiling twists the dublex into
  a tightly interwound superhelix.
• DNA Gyrase introduce (-) supercoiling.

• Eukaryotes
• DNA is wrapped around histone octamers
• to form solenoidal (-) supercoils.

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Condensation
Q What will happen if you remove the histone
core?
Plectonemic supercoils
Solenoidal (Toroidal) supercoils
A The solenoidal supercoil will adopt a
plectonemic conformation
Q How Does Eukaryotic DNA Become Negatively
Supecoiled?
A DNA wrapping around histone cores leads to net
negative supercoils!
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The role of topoisomerases in transcription

• Initiation
• Promotion of helix opening by negative
  supercoiling
• Elongation
• Requirement for topoisomerases to remove ()
  supercoils ahead of the transcription machinary

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Transcription - twin domains
Free rotetion can not occur in vivo
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Transcription - twin domains
Topo I relaxes excess (-) supercoils
DNA Gyrase relaxes () supercoils
Eukaryotes topo I removes both () (-)
supercoils
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DNA topoisomerases as repair enzymes
DNA topoisomerases regulate the organization of
DNA. In addition, they modulate the cellular
sensitivity toward a number of DNA damaging
agents. Increased topoisomerase II activities
contribute to the resistance of both nitrogen
mustard-and cisplatin-resistant cells. Similarly,
cells with decreased topoisomerase II levels show
increased sensitivity to cisplatin, carmustine,
mitomycin C and nitrogen mustard. Topoisomerases
may be involved in damage recognition and DNA
repair at several different levels including
1) the initial recognition of DNA lesions2)
DNA recombination3) regulation of DNA structure.
 
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Topo II specific inhibitors