Campbell Biology – Chapter 16: The Molecular Basis of Inheritance

Life’s Operating Instructions

• 1953: James Watson & Francis Crick publish double-helix model of deoxyribonucleic acid (DNA)
  • DNA encodes hereditary information and is reproduced in every cell cycle
  • Governs biochemical, anatomical, physiological and partially behavioural traits

Early 20th-Century Question: What Is the Genetic Material?

• T. H. Morgan’s chromosomal theory positioned DNA & protein as candidates
• Key model organisms/tools used for discovery
  • Bacteria (​Streptococcus pneumoniae​) & bacteriophages (viruses that infect bacteria)

Griffith’s Transformation Experiment (1928)

• Two ​S. pneumoniae​ strains
  • S (smooth, pathogenic) & R (rough, non-pathogenic)
• Observations
  • Heat-killed S + live R → mice die; live S recovered
• Concept introduced: Transformation = heritable change caused by assimilation of foreign DNA

Avery–MacLeod–McCarty (1944)

• Systematically removed macromolecules from Griffith extracts
• Only destruction of DNA prevented transformation → DNA identified as "transforming substance"
• Skepticism persisted because protein chemistry was better understood than DNA chemistry

Hershey–Chase Blender Experiment (1952)

• Phage T2 labelled with
  • ^{35}!S in protein coat
  • ^{32}!P in DNA
• After infection & blender agitation
  • ^{32}!P entered bacterial pellet → DNA carries genetic program

Chargaff’s Rules (1950)

• Base composition varies between species
• Within any species: #A = #T and #G = #C
• Diversity + regular pairing pattern strengthened DNA’s candidacy

Rosalind Franklin’s X-Ray Diffraction (1952)

• Photo 51 → helical molecule, 2\,\text{nm} diameter, repeating pattern every 0.34\,\text{nm}; one full turn 3.4\,\text{nm} (≈10 bases)
• Suggested two antiparallel sugar-phosphate backbones with nitrogenous bases inside

Watson–Crick Double-Helix Model (1953)

• Antiparallel orientation (5′→3′ vs 3′→5′)
• Specific base pairing dictated by uniform width
  • Purine (A or G) + Pyrimidine (T or C)
  • Hydrogen-bonding rules: A\,\leftrightarrow\,T (2 H-bonds); G\,\leftrightarrow\,C (3 H-bonds)
• Explains Chargaff’s equivalences
• Immediately hinted at a template mechanism for replication

Models of DNA Replication

• Conservative: parent strands re-pair; both strands of daughter new
• Semiconservative (Watson–Crick prediction): each daughter helix = 1 parental + 1 new strand
• Dispersive: mixture of old & new within each strand
• Meselson–Stahl (1958)
  • Grew ​E. coli​ in ^{15}!N, then shifted to ^{14}!N
  • 1st generation hybrid band, 2nd generation hybrid + light → semiconservative confirmed

Origins of Replication & Replication Bubbles

• Prokaryotes: single circular DNA; one origin
• Eukaryotes: hundreds–thousands of origins per linear chromosome
• Replication forks: Y-shaped ends of bubble where DNA is unwound & synthesized

Key Enzymes & Proteins (see also Table 16.1)

• Helicase: unwinds helix
• Single-strand binding proteins (SSB): stabilize separated strands
• Topoisomerase: relieves torsional strain ("overwinding") ahead of fork
• Primase: synthesizes short RNA primers (≈5–10 nt) because DNA polymerases need a 3′-OH
• DNA Polymerase III (prok.) / δ & ε (euk.): main elongation; rate ≈ 500 nt·s⁻¹ (bacteria) or 50 nt·s⁻¹ (humans)
• DNA Polymerase I (prok.) / RNase H + Pol δ (euk.): removes RNA primers & fills gaps with DNA
• DNA Ligase: seals nicks; joins Okazaki fragments & primer-replacement patches
• Sliding Clamp & Clamp Loader: keep polymerase attached (high processivity)

Nucleotide Substrates & Energy

• Incoming monomer = deoxynucleoside triphosphate (dNTP)
  • Example: dATP vs ATP differs by sugar (deoxyribose vs ribose)
• Polymerization releases PP₁ (pyrophosphate) → hydrolysis drives reaction energetically forward

Antiparallel Elongation: Leading vs Lagging Strands

• Polymerases synthesize only 5′→3′
• Leading strand: continuous synthesis toward fork
• Lagging strand: discontinuous synthesis away from fork → short \approx 100–200 nt (euk.) or 1–2 kb (prok.) Okazaki fragments
  1. Primase adds RNA primer for each fragment
  2. DNA pol III extends fragment until it reaches previous primer
  3. DNA pol I replaces RNA with DNA
  4. Ligase seals sugar-phosphate backbone

Replication Factory Concept

• Multiple enzymes form stationary "DNA replication machine"
• Parental DNA is reeled in; daughter strands are extruded as loops

Proofreading & DNA Repair

• DNA pol has 3′→5′ exonuclease activity → immediate correction lowers error to 10^{-7}–10^{-8} per base
• Mismatch repair: post-replicative enzymes excise & resynthesize mispaired region
• Nucleotide Excision Repair (NER) (e.g., thymine dimers from UV)
  1. Nuclease cuts damaged strand
  2. DNA pol fills in
  3. Ligase closes nick
• Persistent errors = mutations, raw material for evolution

End-Replication Problem & Telomeres

• Removal of final primer on lagging strand leaves 5′ overhang → progressive shortening each S phase
• Telomeres: repetitive, non-coding sequences (e.g., \text{TTAGGG}_n in humans) buffer gene loss
• Telomerase (ribonucleoprotein reverse transcriptase)
  • Active in germ cells, early embryos, and many cancer cells
  • Adds telomeric repeats using its own RNA template ⇒ compensates shortening
• Telomere shortening implicated in ageing; telomerase reactivation associated with tumorigenesis

Chromosome Architecture

• Prokaryotic DNA: circular, double-stranded, supercoiled; localized to nucleoid (no membrane)
• Eukaryotic DNA + protein = chromatin; packing hierarchy (diameter references):
  1. DNA double helix – 2\,\text{nm}
  2. Nucleosome (DNA wrapped ~1¾ turns around octamer of histones H2A, H2B, H3, H4) – "beads-on-a-string" 10\,\text{nm} fiber; linker histone H1 anchors
  3. Zig-zag/solenoid 30 nm fiber (H1-mediated folding)
  4. Looped domains attached to protein scaffold – 300\,\text{nm}
  5. Metaphase chromosome – each chromatid \approx 700\,\text{nm}; entire replicated chromosome \approx 1,400\,\text{nm} wide

Functional States of Chromatin

• Euchromatin: loosely packed, transcriptionally active
• Heterochromatin: highly condensed (centromeres, telomeres); transcriptionally silent; difficult for machinery to access
• Histone modifications (acetylation, methylation, phosphorylation) & DNA methylation dynamically regulate compaction and gene expression
• Interphase chromosomes occupy discrete "territories" to avoid entanglement

Key Numbers & Facts to Memorise

• Helix diameter: 2\,\text{nm}; base spacing: 0.34\,\text{nm}; one turn: 3.4\,\text{nm} (≈10 bp)
• Replication speeds: \sim 500 nt·s⁻¹ (bacteria) vs \sim 50 nt·s⁻¹ (human)
• Error rates: raw polymerase \sim 10^{-5}; after proofreading/repair \sim 10^{-10}
• Okazaki fragment size: \approx 1{-}2 \times 10^{3} nt (E. coli) / \approx 100{-}200 nt (euk.)

Connections & Implications

• Central Dogma: Accurate replication underpins transcription & translation fidelity
• Biotechnology: Transformation principle paved way for recombinant DNA & plasmid cloning
• Medicine: Chemotherapeutic drugs target topoisomerase, DNA pol, or telomerase; mismatch-repair defects → Lynch syndrome; NER defects → Xeroderma pigmentosum
• Evolution: Mutation balance (damage vs repair) generates genetic variation for natural selection

Experiment & Concept Map (Quick Review)

• Griffith ➔ transformation
• Avery/MacLeod/McCarty ➔ DNA = transforming principle
• Hershey–Chase ➔ viral DNA enters cell, directs infection
• Chargaff ➔ species specificity + base parity
• Franklin/Wilkins ➔ helical parameters
• Watson–Crick ➔ structure & replication mechanism
• Meselson–Stahl ➔ semiconservative replication proven