DNA Structure & Function 6 September 2010

8/8/2019 DNA Structure & Function 6 September 2010

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DNA Structure & Function

Double helix structure of DNA was proposed in 1953 by James Watson and Francis Crick

Rosalind Franklins DNA image

Microbial Genetics ht10

DNA structure

1. primary base sequence

2. secondary - helical form

forces that hold it together

environmental factor effects

alternative helices & structures

supercoiling

- local structure

static (intrinsic) structure

dynamic structure

3. tertiary structure / protein binding

4. Protein-nucleic interactions where, what, how?

5. Amino acid recognition of bases

6. DNA-binding motifs / DNA distortion upon protein binding7. Wet lab techniques identifying DNA and proteins that interact


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DNA has a phosphodiester backbone Structure of a nucleotide Principle bases in nucleic acid

RNA versus DNA

Features of DNA nucleotides


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1. Primary DNA structure. Base sequence

Chargaffs rules:

1. First parity rule: for duplex DNA %A %T and %G %C (Watson-

Crick base pairing)

2. Cluster rule: bases cluster non-randomly

Base clustering is often related to direction of transcription. The

template strand tends to be pyrimidine rich. The mRNAsynonymous strand tends to be purine rich

Y = pyrimidines

R = purines

3. Second parity rule: %R %Y also in single DNA strands

Associated with ability of DNA and RNA to form stem structures

Loops in RNA tend to be purine rich - May reduce probability of

dsRNA formation between mRNAs (Part of a defence

mechanism against foreign DNA?). Later lecture we will discussregulation by small antisense RNAs

4. The GC rule: (G+C%) is species specific

Codon choice

Mutation pressure

Thermophilicity?

DNA sequence is not random Erwin Chargaff

1.

2.


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2. DNA secondary structure helical form

B-DNA: most common physiological form, right-handed helix, 10 bp per helical turn

A-DNA: RNA/DNA & RNA/RNA helices, DNA at low ionic strength, 11 bp per helical turn

Z-DNA: alternating R & Y or methylated Cs, left-handed helix, 6 bp per helical turn

Triple-helical DNA: polyR, polyY, polyY

B DNA the classic double helix

Hydrogen bonding A-T or G-C holds the two strands together

A-T = 2 H bondsG-C = 3 H bonds


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Does left-handed Z-DNA have any

biological purpose?

Z DNA commonly forms near

transcription start sites.

Z-DNA regions are stabilized by the

negative supercoiling generated bytranscription:

Suggests Z DNA is associated with

transient localized conformational

changes.

Certain classes of proteins bind to Z-

DNA with high affinity and

specificity. Indicates a biological

role.

Still very little known about Z-DNAs

role or significance!

Z DNA Alternating R & Y or methylated Cs, left-handed helix, 6 bp per helical turn


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Intermolecular triplexes = Y:RY, or R:RY (TFO = triplex forming oligonucleotides)

Intramolecular triplexes = H-DNA (occurs naturally in supercoiled DNA)

Intermolecular triplex Intramolecular triplex

H-DNA (intramolecular triplex DNA).PolyRPolyY tract with mirror repeat symmetry, one of the

single strands (shown in blue) folds back and forms a triplex

structure and the other strand (yellow) is left unpaired.

A DNA triplex is formed when pyrimidine or

purine bases occupy the major groove of theDNA double Helix forming Hoogsteen pairs

with purines of the Watson-Crick basepairs.

TFOs are being investigated as gene-drugs, to

modulate gene activity in vivo.

Triple helical DNA = Triplex DNA polyR, polyY, polyY

Watson-Crick base

pairing is illustratedby dotted lines.

Hoogsteen base

pairing by broken

lines
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In addition to the helical coiling of the strands to form a double helix,

the double stranded DNA molecule can also twist upon itself.

Negatively supercoiled DNA is underwound (favors unwinding of the helix)

DNA isolated from cells is always negatively supercoiled

Positively supercoiled DNA is overwound

Supercoiling of DNA

Relaxed DNA has no supercoils

(10.4 bp per turn in B-DNA)

L = linking number = number of times one DNA strand winds in a right-handed direction around the other in the molecule.

Topological property (cannot change without breaking a covalent bond topoisomerase activity).

T = twist = The number of complete revolutions that one DNA strand makes around the duplex axis.

Geometric property (can change by physical deformation of the molecule, without breaking any covalent bonds).

W = writhing number = the number of times the duplex axis turns around the superhelical axis.

= superhelical density = number of supercoils per turn 0.05 in DNA in vivo.


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Topoisomerases

Type I topoisomerases:

Relax negatively supercoiled DNA by nicking then closing one strand of duplex.

Cuts one strand of the double helix, passes the other strand through, then rejoins the cut ends.

Type II topoisomerases: breaks both strands and changes the linking number in steps of2.e.g. DNA Gyrase: introduces negative supercoiling.

Negatively supercoiled DNA = Undertwisted DNA

Easier for proteins to access bases

Easier to separate strands (replication, transcription).

Supercoiling of DNA


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minor

major

Local secondary structure of DNA


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Local secondary structure of DNA

DNA-Protein Interactions often occur in Minor and Major Grooves

minor

major

The number and type on

possible DNA protein

interactions depends on base

sequence and also the major

and minor grooves of DNA


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Tertiary structure of DNA protein binding

Proteins interact with DNA during: replication (recognition oforiand ter)

transcription (promoters, operators, terminators)recombination (homologous & site specific)

defence (restriction enzymes)

Proteins interact with: bases (but these are buried inside the helix)

structurephosphodiester backbone

Protein interactions with DNA involve:

hydrogen bonds between aa side chains and bases or phosphate: 51%

van der Waals interactions (molecular fit) 22%

hydrophobic interactions (aromatic aa to sugar) 19%

electrostatic interactions (salt bridges) 8%

interactions mediated by water (aa to base or phosphate)


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One typical contact of

Protein and DNA

Protein binding


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Protein can bind the DNA through the base, sugar, and the phosphate groups

Hydrogen bonds with phosphate are not specific, but are important in stabilizing

protein-DNA complexes

Guanine exposes the greatest number of potential hydrogen-bonding atoms on

the base edge (4 positions)

Polar and charged residues of amino acids play a central role in DNA binding

Arg > Lys > Ser > Thr; Asn and Gln

Acidic residues are used sparingly Asp and Glu

Relatively few interactions are produced by hydrophobic residues

Protein binding

Example of an Arginine interaction with Guanine


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Distribution of single hydrogen bonds with DNA-bases


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Two main classes of recognition:

Base readout and Shape readout, which are further subdivided as illustrated.

Types of protein-DNA recognition mechanisms used for specificity.


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Sequence-specific patterns on the edges of the bases in the major groove underlie the ability of

proteins to readout base pairs through hydrogen bonds and hydrophobic contacts (hydrogen bond

acceptors in red, donors in blue, thymine methyl group in yellow, and base carbon hydrogens in white).

Note that A:T versus T:A and C:G versus G:C are indistinguishable in the minor groove.

The three panels show successive rotations of 90 around the helix axis.

Base recognition in the major and minor groove.


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Common Motifs in DNA binding proteins:

1. Helix-turn-Helix

2. Zinc Finger

3. Leucine Zipper

4. Helix-loop-Helix


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Examples of Helix-turn-Helix DNA-Binding Proteins


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Example of a Helix-turn-Helix Binding Protein


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Homeodomain Protein in Drosophila utilizing helix-turn-helix motif


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Example of a Helix-turn-Helix Binding Protein - repressor (also in P22)

Helix-2 lies in the major groove of its

DNA target

Critical amino acid residues in the recognition helix

are positioned to facilitate hydrogen bonding with

the edges of base pairs in the DNA


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* are amino acids facing one side of the helix

*

*

* *

The recognition helix of the phage repressor


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Details of-Repressor Binding to

Operator

The helix-turn-helix motif is

inserted into the major groove of

the DNA

The arm of the lower monomer

reaches around to embrace

the DNA



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Geometry of the

repressor-operator complex

1. Recognition helices: 3 & 3

2. Protein-protein interactionhelices: 5 & 5

3. Bending of DNA.


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Amino terminal of Repressor + DNA Details of hydrogen bonds:

base pair 2

base pair 4

base pair 6


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Amino-acid DNA BackboneInteractions


hydrogen bonds form

between peptide NH

groups and phosphates


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Changes of DNA conformation associated with repressor binding

DNA alone Shape when repressor is bound


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Zinc Finger DNA binding protein domain Utilizing a zinc in the center ofan alpha helix and two beta sheets


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DNA-binding by a Zinc Finger Protein Three zinc-fingers forming arecognition site

Alpha helix amino acids in each zinc finger interacts with DNA bases


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Zinc Finger: beta-sheet amino acids can also recognize DNA

A dimer of the zinc finger domain of the glucocorticoid receptor bound to its

specific DNA sequence.


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CS 6463: (P) Control of Gene Expression 32

Zinc fingers: Structure alone does not detect binding Site three examples

The specific amino acids in the

C-terminal alpha helix of the

zinc finger motif determinethe interaction specificity of

DNA recognition


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Helix-loop-helix and Leucine Zippers

Heterodimerization of a Leucine Zipper

Leucine Zipper Dimer

The motif mediates both DNA binding and protein dimerization

Homodimers and heterodimers can recognize

different DNA patterns


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Helix-Loop-Helix (HLH): Helix-loop-helixHelix-loop-Helix motif and its dimer

The helix-loop-helix motif consists of a short alpha helix connected by a loop to alonger alpha helix. Part of this motif is a dimerization domain that interacts with other

helix-loop-helix proteins to form homo- or heterodimers; the dimerization partner

often determines DNA binding affinity and specificity because two alpha-helices, one

from each monomer, bind to the major groove of the target DNA


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Helix-Loop-Helix (HLH): Helix-loop-helix

Truncation of a HLH tail (DNA binding domain)

inhibits binding


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Wet-lab Techniques: Gene Mobility Shift Assay

One can identify the sizes of proteins associated/bound with the desired DNA fragment


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Wet-lab Techniques:DNA Affinity Chromatography

After obtaining the protein, run mass spectroscopy, identify the amino acid sequence, check

against the genome, identify the gene sequence


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Wet-lab Techniques: Detecting DNA Binding Sites

Assay to determine the gene sequence

recognized by a specific proteinChromatin Immunoprecipitation

In vivo genes bound to a known protein

DNA Structure & Function 6 September 2010

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