Molecular Biology: DNA Structure and Packaging

Introduction to DNA Structure and Packaging

• Instructor: Chris Della Vadova, Topic Coordinator for Molecular Biology.
• Lecture Context: This is the first of three lectures for Week 2, focusing on the foundational properties of DNA.
• Core Objective: To understand the chemistry and organization of DNA, which is essential for studying replication, repair, and mutation.
• Lecture Topics Overview:
  • Scientific discovery of DNA as genetic material.
  • Chemistry of the double helix.
  • The scale problem: Fitting DNA into the cell.
  • DNA packaging mechanisms.

Historical Journey: Identifying the Genetic Material

• The Gene Concept: Prior to molecular discovery, Gregor Mendel established the concept of units of inheritance (genes), but the physical substance of genes remained unknown.
• Friedrich Miescher (1869):
  • First to isolate DNA from the nuclei of white blood cells.
  • Used an alkali lysis technique.
  • Named the substance "nuclein."
  • Identified it as a phosphorus-rich substance, though its function was unknown.
• Frederick Griffith (1928) - The Transforming Principle:
  • Organism: Streptococcus pneumoniae (causes pneumonia).
  • Strain Types:
    • R strain: Non-virulent (rough appearance, mouse survives).
    • S strain: Virulent (smooth appearance due to a protein coat, mouse dies).
  • The Experiment:
    1. Infect mouse with Heat-Killed S strain $\rightarrow$ Mouse lives.
    2. Infect mouse with Heat-Killed S strain + Live R strain $\rightarrow$ Mouse dies.
  • Discovery: Live S strain bacteria were recovered from the dead mouse. Griffith concluded that the R strain was transformed into the virulent S strain by a "transforming principle" from the dead S cells.
• Avery, MacLeod, and McCarty (1944):
  • Attempted to identify the chemical identity of Griffith's transforming principle.
  • They treated the S strain extracts with specific enzymes to destroy candidate molecules:
    • Protease: Transformation still occurred (Protein is not the genetic material).
    • RNase: Transformation still occurred (RNA is not the genetic material).
    • DNase: Transformation did NOT occur.
  • Conclusion: DNA is the transforming principle.
• Hershey and Chase (1952) - The Blender Experiment:
  • Used bacteriophages (viruses that infect bacteria) to determine which component (protein or DNA) entered the cell to direct viral production.
  • Used radioactive isotopes:
    • $^{35}S$ (Sulfur): Found only in proteins. Labeled the phage protein coat.
    • $^{32}P$ (Phosphorus): Found only in DNA. Labeled the phage genome.
  • Results: After infection and agitation in a blender to remove "phage ghosts," only radioactivity from $^{32}P$ was found inside the bacterial cells.
  • Conclusion: DNA is the genetic material.

The Double Helix: Models and Chemistry

• Erwin Chargaff's Rules (Base Composition):
  • Analysed base composition across species.
  • Rule 1: The amount of Adenine ($A$) equals Thymine ($T$).
  • Rule 2: The amount of Guanine ($G$) equals Cytosine ($C$).
  • While specific ratios ($AT$ vs $GC$) vary by species (e.g., humans have higher $AT$ content than $GC$), the equal pairing ratios remain constant.
• X-Ray Diffraction Data (King's College London):
  • Rosalind Franklin and Maurice Wilkins: Used X-ray diffraction on DNA fibers.
  • Photograph 51: Franklin's iconic image revealed an "X" shape, characteristic of a helix.
  • Key Measurements: Identified a $3.4\text{Å}$ (Angstrom) rise per base and a $34\text{Å}$ pitch (though the lecture later references a $36\text{Å}$ pitch for B-DNA).
• Watson and Crick (Cambridge, 1953):
  • Developed the structural model of DNA.
  • Synthesized Chargaff's ratios with Franklin's X-ray data (obtained by Wilkins without Franklin's consent).
  • Proposed a base-paired, anti-parallel double helix.
  • Nobel Prize: Awarded to Watson, Crick, and Wilkins; Franklin was post-humously ignored.

Anatomy of a Nucleotide and DNA Polarity

• Nucleotide Components:
  1. Phosphate Group.
  2. Deoxyribose (Pentose Sugar): A five-carbon sugar.
  3. Nitrogenous Base: Attached at the 1’ carbon.
• Nitrogenous Bases:
  • Purines: Double-ring structure (Adenine and Guanine).
  • Pyrimidines: Single-ring structure (Cytosine, Thymine in DNA, and Uracil in RNA).
• Polarity and Phosphodiester Backbones:
  • Every strand has a free 5’ phosphate end and a free 3’ hydroxyl ($OH$) end.
  • Polarity is defined as 5’ to 3’.
  • Processes like replication, transcription, and repair are strictly directional.
• Hydrogen Bonding and Stability:
  • $A$ and $T$ are held together by $2$ hydrogen bonds.
  • $G$ and $C$ are held together by $3$ hydrogen bonds.
  • $GC$ pairs are more thermally stable than $AT$ pairs, impacting PCR primer design and DNA melting points.
• Anti-parallel Alignment: The two strands of the helix run in opposite directions (one 5’ to 3’, the other 3’ to 5’).

Biological Properties of DNA

• B-DNA (Physiological Form):
  • Diameter: $20\text{Å}$.
  • Rise per Base Pair: $3.4\text{Å}$.
  • Turn Pitch: Approximately $36\text{Å}$.
  • Base Pairs per Turn: Approximately $10$.
• Major and Minor Grooves:
  • Major Groove: Wider; exposes more of the base pair edges. Most sequence-specific proteins (transcription factors, restriction enzymes) bind here.
  • Minor Groove: Narrower; has specific binding proteins but is generally less accessible for sequence reading.
• The C-Value Paradox:
  • Genome size does not correlate with organismal complexity.
  • Examples:
    • Lungfish: $130 \times 10^9$ base pairs.
    • Human: $3.2 \times 10^9$ base pairs.
    • Fruit Fly: $140 \times 10^6$ base pairs.
    • Yeast: $12 \times 10^6$ base pairs.
    • Bacteria: $5 \times 10^6$ base pairs.

DNA Packaging and Compaction

• The Scale Problem: The human genome contains approximately $3.2$ billion base pairs. Stretched out, this DNA is $2\text{ meters}$ long, yet must fit into a nucleus only $6\text{ μm}$ wide. This requires a compaction ratio of approximately $6,000,000:1$.
• Nucleosomes - "Beads on a String":
  • Structure: $147$ base pairs of DNA wrapped $1.65$ times around a histone octamer.
  • Histone Octamer: Composed of two copies each of four core Histone proteins: $H2A$, $H2B$, $H3$, and $H4$.
  • Linker DNA: Short segments ($20$ to $80$ base pairs) connecting nucleosomes.
  • Histone H1: A fifth histone that helps maintain the structure of the nucleosome.
  • Conservation: Histones are highly evolutionarily conserved (e.g., $H4$ differs by only two amino acids between peas and cows).
• Histone Modifications (Epigenetics):
  • Histones have N-terminal tails that protrude and can be chemically modified to regulate DNA accessibility.
  • Acetylation: Neutralizes the positive charge of histones, loosening the bond with the negatively charged DNA and allowing for transcription.
  • Methylation: Complex effect; can either increase or decrease chromatin condensation depending on the residue and number of methyl groups.
  • Phosphorylation: Often associated with chromosome condensation and DNA damage responses.

Higher-Order Organization

• Chromatin States:
  • Euchromatin: Reduced condensation; transcriptionally active.
  • Heterochromatin: Highly condensed; transcriptionally inactive.
• Topologically Associating Domains (TADs):
  • Large segments of DNA organized into loops.
  • Maintained by the binding of CTCF (CCTC-binding factor).
• Structural Landmarks of Chromosomes:
  • Centromeres: Regions (usually near the middle) where kinetochore proteins attach, linking chromosomes to spindle microtubules during cell division.
  • Telomeres: Repetitive DNA sequences at chromosome ends that protect against material loss.
    • Telomerase: Enzyme that extends telomeres. Active in germ/stem cells; silenced in most somatic cells but reactivated in many cancers.
• Summary of Compaction Sizes:
  • Helix diameter: $2\text{ nm}$.
  • Nucleosome ("Bead"): $11\text{ nm}$.
  • Packed Chromatin: Hundreds of nanometers to several microns.
  • Metaphase Chromosome: Fully condensed width of $1.4\text{ μm}$ ($1,400\text{ nm}$). Total compaction is about $10,000$-fold.

Key Takeaways

• Scientific discovery progressed from Griffith (transformation) to Avery (DNA principle) to Hershey/Chase (confirmatory virus experiment).
• Structural biology relies on base-pairing rules ($A=T$, $G=C$) and anti-parallel orientation.
• DNA packaging is not just a storage solution but an active regulatory mechanism for gene expression via chromatin state and loops (TADs).