Part 2 DNA Topology Some of the following slides and text are taken from the DNA Topology lecture from Doug Brutlags January 7 2000 Biochemistry 201 Advanced Molecular Biology Course at Stanford University ID: 515795
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Slide1
DNA Lecture
Part 2Slide2
DNA
Topology
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 UniversitySlide3
What Is Supercoiling & Why Should I Care?
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 Slide4
DNA Topology
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 DNASlide5
DNA that is
underwound
is referred to as negatively supercoiled
The helices wind about each other in a
right-handed path
in space
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
spaceSlide6
DNA topologySlide7
Linking number
- # times would have to pass
cccDNA strand through the other to entirely separate the strands and not break any covalent bondsTwist - # times one strand completely wraps (# helical turns) around the other strandWrithe
– when long axis of double helix crosses over itself (causes torsional stress)Slide8
Linking Defined
Linking number, L
k
,
is the total number of times
one strand
of the DNA helix
is linked with the other
in a
covalently closed circular moleculeSlide9
The linking number is only defined for covalently closed DNA and its value is fixed as long as the molecule remains covalently closed.
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.
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
.
L
k
is always an integer since two strands must always be wound about each other an integral number of times upon closure.Slide10
Linking Number, Twists
and WritheSlide11
DNA tied up in knots
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
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.jpgSlide12
Knots, Twists,
Writhe and
Supercoiling
Circular DNA chromosomes, from viruses for instance, exist in a highly compact or folded conformationSlide13
Twist
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)]Slide14
Writhe
Writhe is
the number of 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 ]Slide15
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 zeroSlide16
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
.Slide17
Writhe of supercoiled DNA
Interwound
ToroidalSlide18
Types of SupercoilsSlide19
SupercoilingSlide20
Negative vs. Positive Supercoiling
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
Slide21
Relaxed
SupertwistedSlide22
Unwinding DNASlide23
ToposomeraseSlide24
Topoisomerase II makes ds breaksSlide25
Topoisomerase I makes ss breaksSlide26Slide27Slide28Slide29
X Ray Images of
SupercolingSlide30
Ability of Uracil To Form Stable Base Pairs Enhances RNA’s Ability To Form Stem-loop StructuresSlide31
Histone Variants
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
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 bindingSlide32
Unwrapping of DNA from nucleosome allows DNA-binding proteins access to their binding sites
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 central sites less accessible than those near the ends decreasing probability of protein binding and hence regulating transcriptional activity
more central
more peripheralSlide33
Nucleosome remodeling complexes
Alter stability of DNA-histone 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 DNASlide34
DNA-binding protein dependent nucleosome positioning
Nucleosomes are sometimes specifically positioned
Keeps DNA-binding protein site in linker region (hence accessible)
Can be directed by DNA-binding 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 associateSlide35
Positioning can be inhibitory
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 Slide36
DNA sequences can direct positioning
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 rareSlide37
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 Slide38
Modification of N-terminal tails
Results in increased or decreased affinity of nucleosome for DNA
Modifications include acetylation, methylation and phosphorylationCombination of modifications may encode information for gene expression (positively or negativelySlide39
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 structuresSlide40Slide41
HAT
Acetylation creates binding
sites for bromo- and chromodomain
protein bindingSlide42
Chromatin remodeling complexes and histone modifying enzymes work together to make DNA more accessibleSlide43
Distributive inheritance of old histones
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 Slide44Slide45
Histone assembly requires chaperones
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 polymeraseSlide46
Nucleotides and
primer:template
junction are essential substrates for DNA synthesis