Download File - Mr. Blaschke`s Science Class

Part 2
Some of the following slides and text are taken from
the DNA Topology lecture from Doug Brutlag’s
January 7, 2000 Biochemistry 201 Advanced
Molecular Biology Course at Stanford University
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DNA forms supercoils in vivo
Important during replication and transcription
Topology only defined for a continuous strand - no strand
breakage
Numerical expression for degree of supercoiling:
Lk = Tw + Wr
L:linking number, # of times that one DNA strand winds
about the others strands - is always an integer
T: twist, # of revolutions about the duplex helix
W: writhe, # of turns of the duplex axis about the
superhelical axis is by definition the measure of the
degree of supercoiling
Supercoiling or writhing of circular DNA is a result
of the DNA being underwound with respect to the
relaxed form of DNA
 There are actually fewer turns in the DNA helix than
would be expected given the natural pitch of DNA
in solution (10.4 base pairs per turn)
 When a linear DNA is free in solution it assumes a
pitch which contains 10.4 base pairs per turn
 This is less tightly wound than the 10.0 base pairs
per turn in the Watson and Crick B-form DNA
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DNA that is underwound is referred to as
negatively supercoiled
 The helices wind about each other in a right-
handed path in space
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DNA that is overwound will relax and
become a positively supercoiled DNA
helix
 Positively coiled DNA has its DNA helices
wound around each other in a left-handed
path in space
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Linking number - # times would have to pass
cccDNA strand through the other to entirely
separate the strands and not break any
covalent bonds
Twist - # times one strand completely wraps
(# helical turns) around the other strand
Writhe – when long axis of double helix
crosses over itself (causes torsional stress)
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Linking number, Lk, is the total number of times
one strand of the DNA helix is linked with the
other in a covalently closed circular molecule
The linking number is only defined for covalently closed DNA
and its value is fixed as long as the molecule remains
covalently closed.
2. The linking number does not change whether the covalently
closed circle is forced to lie in a plane in a stressed
conformation or whether it is allowed to supercoil about
itself freely in space.
3. The linking number 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.
4. Lk is always an integer since two strands must always be
wound about each other an integral number of times upon
closure.
1.
Metabolic events involving
unwinding impose great
stress on the DNA because of
the constraints inherent in
the double helix
 There is an absolute
requirement for the correct
topological tension in the
DNA (super-helical density) in
order for genes to be
regulated and expressed
normally
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 For example, DNA must be
unwound for replication and
transcription
Figure from Rasika Harshey’s lab at UT Austin showing
an enhancer protein (red) bound to the DNA in a
specific interwrapped topology that is called a
transposition synapse.
www.icmb.utexas.edu/.../47_Topology_summary.jpg
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Circular DNA chromosomes, from viruses for
instance, exist in a highly compact or folded
conformation
 The linking number of a
covalently closed circular DNA
can be resolved into two
components called the twists,
Tw and the writhes, Wr.
 Lk = Tw + Wr
 The twists are the number of
times that the two strands
are twisted about each other
 The length and pitch of DNA in
solution determine the twist.
[Tw = Length (bp)/Pitch
(bp/turn)]
 Writhe is the number of
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times that the DNA helix
is coiled about itself in
three-dimensional space
The twist and the linking
number, determine the
value of the writhe that
forces the DNA to assume
a contorted path is space.
[Wr = Lk - Tw ]
Unlike the Twist and the Linking number,
the writhe of DNA only depends on the
path the helix axis takes in space, not
on the fact that the DNA has two
strands
 If the path of the DNA is in a plane, the
Wr is always zero
 If the path of the DNA helix were on the
surface of a sphere (like the seams of a
tennis ball or base ball) then the total
Writhe can also be shown to be zero
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Molecules that differ by one unit in linking
number can be separated by electrophoresis in
agarose due to the difference in their writhe
(that is due to difference in folding).
 The variation in linking number is reflected in a
difference in the writhe.
 The variation in writhe is subsequently reflected
in the state of compaction of the DNA
molecule.
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Interwound
Toroidal
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Right handed supercoiling =
negative supercoiling
(underwinding)
Left handed supercoiling =
positive supercoiling
Relaxed state is with no
bends
DNA must be constrained:
plasmid DNA or by proteins
Unraveling the DNA at one
position changes the
superhelicity
Relaxed
Supertwisted
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Alter nucleosome function
 H2A.z often found in areas
with transcribed regions of
DNA
 prevents nucleosome from
forming repressive structures
that would inhibit access of
RNA polymerase
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Mark areas of chromatin
with alternate functions
 CENP-A replaces H3
 Associated with nucleosomes
that contain centromeric DNA
 Has longer N-terminal tail
that may function to increase
binding sites available for
kinetochore protein binding
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Many DNA-binding proteins
require histone-free DNA
DNA-histone interactions
dynamic: unwrapping is
spontaneous and
intermittent
Accessibility to binding
protein sites dependent on
location in nucleosomal DNA
more peripheral
 more central sites less accessible
than those near the ends
decreasing probability of protein
binding and hence regulating
transcriptional activity
more central
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Alter stability of DNAhistone interaction to
increase accessibility of
DNA
Change nucleosome
location
Require ATP
3 mechanisms:
Slide histone octamer
along DNA
Transfer histone octamer
to another DNA
Remodel to increase
access to DNA
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Nucleosomes are sometimes
specifically positioned
Keeps DNA-binding protein
site in linker region (hence
accessible)
Can be directed by DNAbinding proteins or by specific
sequences
Usually involves competition
between nucleosomes and
binding proteins
If proteins are positioned such
that less than 147 bp exists
between them, nucleosomes
cannot associate
Some proteins can bind
to DNA and a
nucleosome
 By putting a tightly
bound binding protein
next to a nucleosome,
additional nucleosomes
will assemble
immediately adjacent to
the protein preferentially
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DNA sequences that
position nucleosomes are
A-T or G-C rich because
DNA is bent in
nucleosomes
By alternating A-T or G-C
rich sequences, can
change the position in
which the minor groove
faces the histone
octamer
These sequences are rare
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Majority of nucleosomes are not positioned
Tightly positioned nucleosomes are usually
associated with areas for transcription
initiation
Positioned nucleosomes can prevent or
enhance access to DNA sequences needed
for binding protein attachment
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Results in increased or decreased affinity of
nucleosome for DNA
Modifications include acetylation,
methylation and phosphorylation
Combination of modifications may encode
information for gene expression (positively or
negatively
Acetylated nucleosomes are associated with
actively transcribed areas because reduces
the affinity of the nucleosome for DNA
 Deacetylation associated with inactive
transcription units
 Phosphorylation also increases transcription
 Like acetylation, phosphorylation reduces
positive charge on histone proteins
 Methylation represses transcription
 Also affects ability of nucleosome array to
form higher order structures
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HAT
Acetylation creates binding
sites for bromo- and chromodomain
protein binding
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Old histones have to be
inherited to maintain
histone modifications
and appropriate gene
expression
H3▪H4 tetramers are
randomly transferred to
new daughter strand,
never put into soluble
pool
H2A▪H2B dimers are put
into pool and compete
for association with
H3▪H4 tetramers
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Assembly of nucleosome
is not spontaneous
Chaperone proteins are
needed to bring in free
dimers and tetramers
after replication fork has
been passed
Chaperones are
associated with PCNA,
the sliding clamp protein
of eukaryotic replication,
immediately after PCNA
is released by DNA
polymerase