DNA Structure and Supercoiling

Steps of Griffith's Transformation Experiment

• Prepare two strains:

  • S strain (smooth) → virulent (capsule)

  • R strain (rough) → non-virulent (no capsule)

• Inject into mice:

  • Live S → mouse dies

  • Live R → mouse lives

  • Heat-killed S → mouse lives

• Critical step:

  • Mix heat-killed S + live R

  • Inject into mouse → mouse dies

• Recover bacteria from dead mouse:

  • Find live S strain bacteria

Interpretation of Griffith's Transformation Experiment

• Something from dead S cells:

  • Survived heat

  • Entered R cells

  • Permanently changed phenotype

Called “transforming principle”:

• Transformation is:

  • Heritable

  • Stable

• Implies transfer of genetic information

Steps of Avery–MacLeod–McCarty Experiment

• Isolate material from heat-killed S bacteria

• Fractionate into:

  • DNA

  • RNA

  • Protein

• Treat samples with specific enzymes:

  • Protease → destroys proteins

  • RNase → destroys RNA

  • DNase → destroys DNA

• Add treated extracts to live R bacteria

Results from Avery–MacLeod–McCarty Experiment

• Protease-treated → transformation occurs

• RNase-treated → transformation occurs

• DNase-treated → NO transformation

Interpretation of Avery–MacLeod–McCarty Experiment

Only DNA destruction stops transformation
➡ DNA = genetic material

Strength and weakness of Avery–MacLeod–McCarty Experiment

• Strength:

  • Highly specific biochemical test

• Weakness (historical skepticism):

  • Possible protein contamination

Steps of Hershey–Chase Experiment

Grow phage in:

• ³²P → labels DNA

• ³⁵S → labels protein

1. Infect E. coli with labelled phage

2. Allow phage to attach and inject material

3. Use blender → removes phage coats

4. Centrifuge:

   • Pellet → bacteria

   • Supernatant → phage coats

Results of Hershey–Chase Experiment

• ³²P (DNA) → found in bacteria + progeny

• ³⁵S (protein) → remains outside (phage ghosts)

Interpretation of Hershey–Chase Experiment

Only DNA enters cell and is inherited
➡ DNA = genetic material

This experiment:

• Directly tracks molecule movement

• Uses clear physical separation

Phosphodiester Bond Formation mechanism

• 3′ OH performs nucleophilic attack on:

  • α-phosphate of incoming nucleotide

• Releases pyrophosphate (PPi)

Drives reaction forward (irreversible in cells)

Nucleotide energy comes from

• Breaking phosphoanhydride bonds

• Same principle as ATP hydrolysis

Structural consequences of nucleotides

• Backbone is:

  • Negatively charged

  • Interacts with:

    • Mg²⁺ ions

    • DNA-binding proteins

Base Pairing Specificity of nucleotides

A–T and G–C pairing ensures:

• Constant helix width

• Accurate replication

Nucleotide Tautomerisation

Example:

• T (keto) → rare enol form

• Can pair with G instead of A

• Leads to Transition mutations

Chargaff experimental steps in discovery of dna structure

• Extract DNA from organisms

• Chemically quantify bases

• Compare ratios

Chargaff’s rules

Amount of purine base = Amount of pyrimidine bases:  [A]+[G]=[C]+[T]

Amount of guanine = Amount of cytosine:    [G]=[C]

Amount of adenine = Amount of thymine:    [A]=[T]

Suggests:

• Base pairing

• DNA carries information

X-ray diffraction experimental steps in discovery of dna structure

• Prepare aligned DNA fibers

• Expose to X-rays

• Measure diffraction pattern

X-ray diffraction observations

• X-shaped pattern → helix

• Spacing:

  • 3.4 Å → base stacking

  • 34 Å → full turn (10bp per turn)

X-ray diffraction interpretations

DNA is:

• Helical - 2 intertwined helices

• Regularly repeating

Watson and Crick model key features

•Two polynucleotide chains wound around each other in a right-handed helix

•Two chains are antiparallel

•Hydrophilic sugar-phosphate backbones on outside, hydrophobic bases on inside

•Bases of two strands held by H-bonds: 3 between G-C, 2 between A-T

•Base stacking contributes significantly to helix stability – sequence dependent

•Bases 0.34 nm apart, one full turn (10.5 bp)=  3.6 nm

•Helix diameter 2 nm

•Because of base H-bonding, the opposite sugar-phosphate backbones not equally spaced – major (info rich) and minor groove

• Purine–pyrimidine pairing Maintains constant width

B-DNA structure

• Two complementary DNA strands are antiparallel

• Two polynucleotide strands wind around each other in a right-handed double helix (clockwise)

• Hydrophilic sugar phosphate backbone is on the outsideof the molecule – hydrophobic bases on the inside

• Base pairs form a stack on the interior of the helix. 

• Van der Waals interactions between bases stabilize the interactions –important for stability of DNA

• Contribution of base stacking to stability varies with sequence – depends on neighbouring bases

• Crystal structure of B DNA (Dickerson dodecamer) characterized in 1980 – confirmed key aspects of Watson and Crick’s model.

• Helix diameter is ~2 nm (1 nm = 10^-9 m)

• 10.5 base pairs in one complete turn of helix

• Base pairs are 0.34 nm apart

• One full turn of helix is 3.57 nm (0.34 nm/bp x 10.5 bp/turn)

• In B-DNA, the helix forms a major groove and a minor groove - govern interactions with other molecules

• B-DNA is the predominant configuration in cells

• Optimised for: Stability, Protein interaction, Information accessibility

Why grooves exist

• Base pairs are not centred symmetrically

• Glycosidic bonds (base–sugar links) are offset

• This creates:

• Major groove (wide)

• Minor groove (narrow)

Major Groove

Information-Rich Surface

Each base pair exposes a unique pattern of functional groups:

• Hydrogen bond donors (d)

• Hydrogen bond acceptors (a)

• Methyl groups (M, e.g. thymine CH₃)

Major groove example

• A–T → different pattern than T–A

• G–C → different pattern than C–G

So:

• Sequence orientation matters

• Each base pair is chemically distinguishable

importance of major grooves

Proteins can “read” DNA without opening it

• Transcription factors bind via:

  • Hydrogen bonding

  • Shape recognition

• They recognise:

  • specific sequences via major groove patterns

Minor groove

• A–T and T–A look the same

• G–C and C–G look the same

No orientation-specific information

A-DNA structure

• Dehydrated / Protein-Induced Form

• Shorter, wider helix (2.6 nm)

• ~11 bp/turn

• Bases tilted away from perpendicular axis

why A-DNA forms

• Low water → backbone collapses inward

• Bases become more tilted and compact

A-DNA occurrence

• DNA–RNA hybrids

• Protein-bound DNA

• Often seen during transcription

Z-DNA structure

• Left-handed helix

• Zig-zag backbone

• 12 bp/turn

• Narrow (1.8 nm)

Why Z-DNA forms

Requires:

• Alternating purine–pyrimidine (e.g. GC repeats)

• Cytosine methylation

• High salt or torsional stress

Z-DNA importance

• Alternating sequence allows backbone to flip orientation

• Reduces torsional strain

• Relieve supercoiling stress

• Mark actively transcribed genes

Supercoiling

DNA behaves like a twisted elastic rod:

• If over- or under-twisted → forms coils

Supercoiling = DNA twisting on itself

Relaxed DNA

Normal helical turns

Negative supercoiled

• DNA is underwound

• Helix is destabilised

• Base pairs easier to separate

• Facilitates: Replication, Transcription, DNA repair

Positive supercoiled

overwound

How supercoiling forms

Starting point:

• Closed circular DNA

• Example: 20 turns = relaxed

Step 1: Break DNA - Cut one strand (or both)

Step 2: Unwind DNA by 2 turns → now 18 turns

Step 3: Reseal DNA - Linking number fixed at new value

Step 4: Compensation

• DNA wants to return to preferred twist (~10.5 bp/turn)

• So it introduces: Writhe (supercoils)

DNA compensates:

• ↓ twist → ↑ writhe

• ↑ twist → ↓ writhe

Non B-DNA structures formed in genomic repetitive sequences

• Cruciform (inverted repeats)

• Slipped (hairpin) structure - direct repeats

• G-Quadruplex DNA (G4 DNA)

supercoiling equations

Lk=Tw+Wr

• Lk (linking number):

  • Total number of strand crossings

  • Fixed unless DNA is cut

• Tw (twist):

  • Number of helical turns in DNA fragment

• Wr (writhe):

  • Number of supercoils - can be +/-

Topoisomerases

• Control DNA topology

1. Bind DNA

2. Cut strand(s)

3. Pass DNA through break

4. Reseal

• Type I → single strand

• Type II → double strand

Prevents:

• DNA tangling

• replication stress

Major Groove + Protein Binding

• Gene regulation

• Sequence recognition

Supercoiling + DNA Activity

• Negative supercoiling:

  • Promotes strand separation

• Essential for:

  • Initiation of transcription

Cruciform

The sequences on the same strand are:

• Palindromic (read similarly in opposite directions)

• Sites for:

  • Recombination

  • Transcription regulation

• Recognised by specific DNA-binding proteins

Cruciform formation

Step 1: Negative supercoiling

• DNA becomes underwound

• Local regions partially unwind

Step 2: Strand separation

• Hydrogen bonds between strands break

Step 3: Intrastrand pairing

• Each strand folds back on itself

• Complementary inverted repeats pair

Step 4: Cruciform extrusion

• Two hairpin loops form

• Structure becomes cross-shaped

Slipped (Hairpin) Structures

• Repeated sequences in the same direction

• Bulges or loops

• Hairpin-like folding possible

• Causes:

  • Repeat expansion

  • Genome instability

Slipped (Hairpin) struture mechanism

Step 1: DNA replication or repair

• Strands temporarily separate

Step 2: Misalignment

• Repeats pair incorrectly:

  • One repeat aligns with another copy

Step 3: Loop formation

• Extra bases form unpaired loops

Step 4: Stabilisation

• Loop becomes “fixed” during replication

Slipped (Hairpin) structure outcomes

• Template strand loop - deletion

• new strand loop - insertion

G-Quadruplex DNA (G4 DNA)

• Sequence Requirement: Oligo(G)n tracts

• Multiple runs of guanine bases

• Very stable

• Can form:

  • In single strand

  • Between multiple strands

• Parallel or antiparallel orientations

• Found in: Telomeres and Promoter regions

• Regulates: Transcription and Replication

Can block polymerases → slows replication/transcription

G-Quadruplex DNA (G4 DNA) formation

Step 1: Strand separation

• Often occurs in:

  • Transcription

  • Replication

Step 2: Guanine alignment

• Four guanines arrange in a square planar structure

Step 3: G-quartet formation

• Held together by Hoogsteen hydrogen bonds

Step 4: Stacking

• Multiple quartets stack → stable column

Step 5: Stabilisation

• Requires:

  • K⁺ or Na⁺ ions in central channel