Week 2 - DNA Replication, Fidelity, Chromosome Architecture & Telomeres

Learning Outcomes ⬇️

  • Lecture 3
    • Demonstrate understanding of the Meselson–Stahl experiment.
    • List/describe all molecular components that cooperate at a replication fork.
  • Lecture 4
    • Explain how DNA-polymerase base-pair recognition + 3′!→!5′3' !\rightarrow ! 5' exonuclease proofreading increase fidelity.
    • Describe eukaryotic chromosome architecture (chromatin → nucleosomes → higher-order fibres).
    • Correlate mitotic phases with centromere behaviour.
    • Explain the end-replication problem & telomerase-mediated solution.

1 Genetic Stability & Mutation Rates 🧬

  • Organisms demand extreme genomic stability.
    • Observed error rate during normal replication: ≈1 mutation per 109 bp\approx 1 \text{ mutation per } 10^{9} \text{ bp} copied.
    • Combined fidelity mechanisms (polymerase selectivity + proofreading + mismatch repair) yield overall accuracy: 1!/10101!/10^{10} (Table 5-1).
  • Implications
    • Enables long-term information storage while permitting occasional variation for evolution.
    • Therapeutic angle: drugs that selectively elevate error rates in viruses/cancer → lethal mutagenesis.

2 Historical Proof of Semiconservative Replication 🧪

2.1 Competing Models (pre-1958)
  • Conservative: parental duplex preserved, daughter duplex entirely new.
  • Semiconservative: each daughter duplex contains 1 parent + 1 new strand.
  • Dispersive: parental segments interspersed within both daughters.
2.2 Meselson–Stahl Experiment (E. coli, 1958)
  • Methodology
    • Grew bacteria on “heavy” 15!N^{15}!N ammonium chloride → uniformly dense DNA.
    • Shifted cells to “light” 14!N^{14}!N medium; harvested DNA after successive generations.
    • Ultracentrifuged extracts in CsCl\text{CsCl} density gradient at 140,000 g140{,}000\,g for 20 h20\,h → DNA bands separated by buoyant density.
  • Predictions
    • 0 generation: single “heavy” band.
    • 1 generation (if semiconservative): single “intermediate” band.
    • 2 generations: equal mix of “light” and “intermediate”.
  • Observed percentages (band intensities)
    • Gen 0 ≈100%\approx 100\% heavy.
    • Gen 1 ≈100%\approx 100\% intermediate.
    • Gen 2 ≈50%\approx 50\% light / 50%50\% intermediate.
    • Gen 3–4 shift progressively toward light (data table supplied on slide 9).
  • Conclusion ➜ replication is semiconservative.
  • Aesthetic/educational impact: often cited as “one of biology’s most beautiful experiments” (videos linked on slide 6).

3 Core Chemistry of DNA Polymerisation ⚙️

  • Directionality: new strand elongated only 5′!→!3′5' !\rightarrow ! 3'.
  • Substrate: dNTPs; incorporation releases PPi\text{PP}_{\text{i}} → hydrolysis provides driving energy.
  • Enzyme: DNA polymerase (hand-shaped domain architecture).
    • “Fingers” clamp around correct base through induced fit.
    • Requires free 3′-OH3'\text{-OH} primer terminus; cannot initiate de novo.
  • Priming: Primase synthesises ∼10!–!20\sim 10!–!20 nt RNA primers.
    • Question posed: Why RNA, not DNA? ➜ misincorporated ribonucleotides mark primers for later removal; prevents permanent preservation of initiation errors; energetically cheaper.
  • Sliding clamp (PCNA/β-clamp): ring-shaped trimer/dimer that tethers polymerase, ↑ processivity.
    • Loaded by ATP-driven clamp loader complex; dissociates after assembly.

4 Replication Origins & Fork Dynamics 🔱

  • Origin of replication (ori)

    • Defined by AT-rich sequences + initiator protein binding motifs.
    • Prokaryotes: single ori per circular chromosome (speed ≈500!–!1000 nt s−1\approx 500!–!1000\,\text{nt s}^{-1}).
    • Eukaryotes: hundreds per chromosome, spaced ∼30!–!300 kb\sim 30!–!300\,\text{kb}; slower forks (≈ 50 nt s−150\,\text{nt s}^{-1}) yet finish on time via parallel firing.
    • Bacterial ori fire only after A methylation status signals “ready” & nutrient status suffices.

  • Unwinding Ensemble

    • Helicase: ATPase that translocates 5’→3’ or 3’→5’ on one strand; unzips duplex up to ∼1000 bp s−1\sim 1000\,\text{bp s}^{-1}.
    • Single-strand Binding Proteins (SSB/RPA): coat ssDNA cooperatively, prevent hairpins & nucleases.
    • Topoisomerase: relieves superhelical tension ahead of fork (not detailed in early slides but listed under “tidying-up”).

  • Leading vs Lagging Synthesis (Figure 13.16)

    • Leading strand: continuous, same direction as fork movement.
    • Lagging strand: discontinuous Okazaki fragments (prokaryotes ≈ 10001000 nt, eukaryotes ≈ 100100 nt).
    • Polymerase loops lagging template to coordinate dimeric activity; new fragment primed, extended, released.
    • Primer removal: RNase H + DNA pol I (prokaryotes) or RPA/FEN1 pathways (eukaryotes) → nick sealed by DNA ligase (ATP-dependent).

5 High-Fidelity Mechanisms 🛡️

  1. Base-selection (energetics & induced-fit) → 1!/1051!/10^{5} errors.
  2. Exonucleolytic Proofreading
    • Polymerase possesses separate 3′!→!5′3' !\rightarrow ! 5' exonuclease site.
    • Mismatched terminal base impedes forward translocation, promotes strand shift into exonuclease pocket → removal → resume synthesis.
    • Explains necessity of net 5′!→!3′5' !\rightarrow ! 3' synthesis (hypothetical opposite direction would stall after excision due to lack of high-energy triphosphate).
  3. Strand-Directed Mismatch Repair (MMR)
    • In bacteria: hemimethylated GATC motifs differentiate old (methylated) vs new (unmethylated) strand; MutS–MutL–MutH effectors.
    • In eukaryotes: transient nicks signal new strand; MutSα/β & MutLα recruit exonucleases.
    • Lowers errors another 102!–!10310^{2} !–! 10^{3}-fold.
  • Medical insight: germline MMR defects → Lynch syndrome (hereditary non-polyposis colorectal cancer).

6 Chromosome Architecture 🧩

6.1 DNA Packaging Challenges
  • Human diploid genome ≈ 6.4 Gb≈2.2 m6.4\,\text{Gb} \approx 2.2\,\text{m} linear length.
  • Must fit inside ∼6 µm\sim 6\,µ\text{m} nucleus ⇒ >10^{5}-fold compaction.
  • Analogy: stuffing 15.4 km15.4\,\text{km} of thread into a golf ball.
6.2 Nucleosome (basic unit)
  • Core octamer = 2×(H2A, H2B, H3, H4); DNA wraps 146146 bp ≈ 1.7 turns.
  • Linker DNA ≈ 5050 bp; H1 binds linker, promotes 30-nm fibre.
  • Histones: rich in Lys/Arg\text{Lys/Arg} → (+) charge facilitates electrostatic wrapping.
6.3 Higher-order Structure
  • “Beads-on-a-string” → 30-nm solenoid / zig-zag fibre → looped domains (300 nm) → fully condensed metaphase chromosome (1400 nm diameter).
  • Dynamic compaction regulated by histone PTMs & remodelers.
6.4 Centromere
  • Single locus per chromosome; contains satellite repeats (human: ∼170\sim 170 bp units, 2 k–30 k copies).
  • Platform for kinetochore assembly; anchors spindle microtubules during mitosis/meiosis.
6.5 Telomere
  • Tandem repeats (human: TTAGGGn\text{TTAGGG}_{n}) + bound “shelterin” protein complex.
  • Forms T-loop; hides free 3′3' overhang from DNA damage sensors.

7 Cell Cycle & Mitosis 🕰️

  • Phases: G<em>1→S→G</em>2→MG<em>1 \rightarrow S \rightarrow G</em>2 \rightarrow M + cytokinesis.
  • S phase: genome duplication (topics above).
  • Mitosis details
    • Prophase: chromatin condensation; nuclear envelope breakdown.
    • Metaphase: chromosomes align at metaphase plate.
    • Anaphase: sister chromatids segregate (centromere splits).
    • Telophase: re-formation of nuclei; cytokinesis.
  • Outcome: two genetically identical diploid daughter cells; maintains chromosome number.

8 End-Replication Problem & Telomerase 🧪

  • Lagging strand cannot replicate extreme 3′ ends after RNA primer removal → progressive shortening (diagram slide 72).

  • Solution: Telomerase (ribonucleoprotein reverse transcriptase)

    1. Internal RNA template base-pairs with 3′ overhang.
    2. Adds telomeric repeats 5′!–!3′5'!–!3' to parental strand (RNA-templated DNA synthesis).
    3. Conventional DNA pol fills complementary lagging strand; resulting terminus still retains protective 3′ overhang.

  • Telomere length acts as “mitotic clock”

    • Somatic cells lack active telomerase → ∼100!–!200\sim 100!–!200 nt lost/division → senescence checkpoint.
    • Stem cells & germline express telomerase → long-term proliferative capacity.
    • Tumours often reactivate telomerase (90 % of cancers) → target for anti-cancer drugs.

  • Clinical illustration: Werner syndrome

    • Autosomal recessive; mutation in WRN helicase component of shelterin.
    • Phenotype: premature ageing, cataracts, alopecia, atrophy; associated with abnormally short telomeres.

9 Key Enzymatic Cast & Their Functions 🎭

  • Helicase (ATPase): strand separation.
  • SSB/RPA: stabilise ssDNA.
  • Primase: RNA primer synthesis.
  • DNA polymerase α/δ/ε (eukaryotes) or Pol III core (prokaryotes): chain elongation.
  • Sliding clamp (PCNA/β): processivity.
  • Clamp loader (RFC/γ-complex): loads clamp using ATP.
  • RNase H + FEN1/Pol I: primer removal.
  • DNA ligase I: nick sealing (ATP or NAD⁺ dependent).
  • Topoisomerase I/II: relieve torsion; decatenate daughter circles.
  • Telomerase: solves end problem.

10 Applications, Analogies & Ethical Angles 🌍

  • Antiviral/Cancer therapeutics: nucleoside analogues (e.g. AZT) exploit polymerase requirement for 3′-OH; chain termination halts viral/cancer DNA synthesis.
  • Epigenetic regulation: nucleosome positioning & histone modifications influence gene expression; links replication with chromatin assembly.
  • Ageing research: telomere length as biomarker; controversial proposals to up-regulate telomerase in somatic tissues → trade-off between rejuvenation and oncogenic risk.
  • Biotechnology: PCR mimics natural replication but uses heat for denaturation; high-fidelity polymerases leverage proofreading mutations for accurate cloning.

11 Clean-Up & Fork Progression Summary 🧹

  1. Unwind: Helicase + Topoisomerase relieve strain; SSB coat ssDNA.
  2. Prime: Primase lays RNA primer.
  3. Elongate: DNA pol synthesises leading continuously, lagging discontinuously.
  4. Processivity: Sliding clamp tethers polymerase.
  5. Primer Removal: RNase H/FEN1 or Pol I.
  6. Fill & Seal: DNA pol δ/ε (euk) fills gaps; ligase seals phosphodiester bonds.
  7. Proofread & Repair: Exonuclease + MMR ensure final error rate ≤10−10\le 10^{-10}.

12 Useful Multimedia (for revision) 📺

  • DNA replication animations:
    • WEHI ‘DNA Replication in Real Time’: https://youtu.be/7Hk9jct2ozY
    • 3D interactive (Wiley): http://tinyurl.com/dna-repl-animation
  • Meselson–Stahl documentaries.
  • Telomerase mechanism clips.

End of comprehensive bullet-note set – covers all major & minor points, mechanisms, numerical data, examples & clinical relevance presented in slides 1-79 of Lectures 3 & 4.