DNA Lecture - PowerPoint Presentation

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DNA Lecture - PPT Presentation

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

number dna linking binding dna number binding linking writhe nucleosome strand protein histone supercoiling nucleosomes helix times proteins topology

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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

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

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

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 breaksSlide26
Slide27
Slide28
Slide29

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

acetylation

, phosphorylation reduces positive charge on

histone

proteins

Methylation represses transcription

Also affects ability of