DNA Lecture Part 2

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 University

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

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 DNA

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 space

DNA topology

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)

Linking Defined

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

1. 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.

Linking Number, Twists and Writhe

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.jpg

Knots, Twists, Writhe and Supercoiling

Circular DNA chromosomes, from viruses for instance, exist in a highly compact or folded conformation

TwistThe linking number of a

covalently closed circular DNA can be resolved into two components called the twists, Tw and the writhes, Wr.

Lk = Tw + WrThe 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)]

WritheWrithe 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 ]

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

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.

Writhe of supercoiled DNA

Interwound Toroidal

Types of Supercoils

Supercoiling

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

Relaxed

Supertwisted

Unwinding DNA

Toposomerase

Topoisomerase II makes ds breaks

Topoisomerase I makes ss breaks

X Ray Images of Supercoling

Ability of Uracil To Form Stable Base Pairs Enhances RNA’s Ability To Form Stem-loop Structures

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 binding

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 peripheral

Nucleosome remodeling complexes

Alter stability of DNA-histone interaction to increase accessibility of DNA

Change nucleosome location

Require ATP 3 mechanisms:1. Slide histone octamer

along DNA2. Transfer histone

octamer to another DNA

3. Remodel to increase access to DNA

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 associate

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

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 rare

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

Modification of N-terminal tails 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 transcriptionLike acetylation, phosphorylation reduces

positive charge on histone proteinsMethylation represses transcriptionAlso affects ability of nucleosome array to

form higher order structures

HAT

Acetylation creates binding sites for bromo- and chromodomain protein binding

Chromatin remodeling complexes and histone modifying enzymes work together to make DNA more accessible

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

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 polymerase

Nucleotides and primer:template junction are essential substrates for DNA synthesis