DNA Replication, Repair & Telomeres – Comprehensive Bullet Notes

Learning Resources & Administrative Details

• Instructor welcomes questions via Elantra discussion forum → encourages active, asynchronous engagement.
• Lecture constructed around posted Learning Objectives.
• Recommended companion texts:
  • Lippincott’s Illustrated Reviews: Biochemistry, 8ᵗʰ ed., Ch 30.
  • Lippincott’s Illustrated Reviews: Cell & Molecular Biology, 3ʳᵈ ed., Ch 7.

Cell-Cycle Context for DNA Replication

• Eukaryotic cell-cycle phases: G₁ → S → G₂ → M.
• S (Synthesis) phase: site of genomic duplication.
• Goal: each daughter cell inherits an identical genome—basis of genetic continuity.
• Cell-cycle checkpoints enforce tight temporal regulation of replication machinery.

Core Terminology (keep at fingertips!)

• DNA replication: generation of 2 identical DNA molecules from 1 parental duplex.
• Daughter DNA: product strands passed to daughter cells.
• Fidelity: accuracy of incorporated nucleotides (error rate ≈ $10^{-8}$ to $10^{-10}$/bp).
• Processivity: # nt added per polymerase-DNA binding event.
• Ori / ORI: origin sequence where replication initiates.
• Replisome: complete protein ensemble required for replication.
• Primosome: subset that synthesises RNA primers (esp. for Okazaki fragments).

Fundamental Properties of DNA Replication

• Semiconservative: each daughter duplex = 1 parental strand + 1 newly synthesised strand.
• Semi-discontinuous:
  • Leading strand → continuous 5′→3′ synthesis.
  • Lagging strand → discontinuous synthesis as Okazaki fragments.
• Bidirectional: replication forks move in both directions away from each Ori.
• Directionality rule:
  • Polymerase extends ONLY $5' \rightarrow 3'$.
  • Template read $3' \rightarrow 5'$ (antiparallel).
• DNA must transiently melt; polymerases require single-stranded template.

Prokaryotic DNA Replication

Initiation @ OriC (circular chromosome)

• DNA helicase: unwinds parental duplex.
• SSB proteins: prevent re-annealing.
• Topoisomerases alleviate positive supercoils:
  • Type I ⟹ single-strand nick.
  • Type II (e.g.
  DNA gyrase) ⟹ double-strand cut; antibiotic targets (ciprofloxacin, levofloxacin).
• Primase (DnaG): RNA primer (~10 nt) supplies free $3'\text{-OH}$.
• Multiple primers on lagging strand; single primer on leading strand.

Elongation Machinery

• DNA Polymerase III holoenzyme (multi-subunit):
  • Highest processivity → ensured by β sliding clamp (ring encircles DNA; ATP-driven clamp loader).
  • Proof-reading via $3' \rightarrow 5'$ exonuclease.
• Catalytic chemistry: nucleophilic attack of $3'\text{-OH}$ on incoming dNTP α-phosphate (Mg²⁺ co-factor), releasing PPᵢ.
• Lagging-strand maturation:
  • DNA Pol I replaces RNA primers (has $5' \rightarrow 3'$ exonuclease + polymerase).
  • DNA ligase seals nick using NAD⁺ (bacteria) or ATP (eukaryotes).

Termination

• TER sequences bind Tus (Terminus Utilisation Substance) protein → replication fork arrest and replisome disassembly.

Eukaryotic DNA Replication (added layers of complexity)

• Genomic scale & nucleosome packaging necessitate multiple ORIs per chromosome.
• Partial list of functional homologues:
  • ORC = Origin Recognition Complex (licensing).
  • MCM = helicase.
  • RPA = eukaryotic SSB.
  • Pol α/primase = hybrid enzyme: RNA primer + ~20 nt DNA “patch”.
  • Pol ε = leading-strand polymerase.
  • Pol δ = lagging-strand polymerase.
  • PCNA = trimeric sliding clamp (proliferation marker in tumours).
  • Primer removal by RNase H + FEN-1 (Flap Endonuclease 1).

Telomeres & Telomerase

• Telomere = tandem repeats (human: $\text{TTAGGG}_n$) + protective proteins (Shelterin).
• End-replication problem: final RNA primer on lagging strand cannot be replaced → progressive 3′ overhang shortening per division.
• Biological outcomes: cellular ageing, senescence, apoptosis.
• Telomerase (ribonucleoprotein):
  • Reverse transcriptase subunit (TERT).
  • RNA template (TERC) complementary to telomeric repeat.
  • Extends 3′ G-rich overhang; pol α then fills C-strand.
• High telomerase activity in germ cells, stem cells, >90 % cancers (therapeutic target).

DNA Repair Pathways (4-step archetype)

1. Damage recognition.
2. Excision of damaged region.
3. Resynthesis by DNA Pol (template-directed, $5' \rightarrow 3'$).
4. Ligation by DNA ligase.

1. Mismatch Repair (MMR)

• Corrects replication errors escaping Pol proof-reading.
• MutS / MutL / MutH (prokaryotes) detect hemimethylated sites (parental strand = methylated).
• Exonuclease removes stretch; Pol III re-synthesises; ligase seals.
• Eukaryotic analogues exist (MSH, MLH) though strand discrimination cues differ.

2. Nucleotide Excision Repair (NER)

• Targets bulky distortions such as UV-induced thymine dimers.
• Prokaryotic UvrA/UvrB/UvrC endonuclease cuts ~12–13 nt segment; Pol I + ligase restore.
• Constitutive throughout cell cycle.
• Genetic defects ⟹ Xeroderma pigmentosum (autosomal recessive, extreme UV sensitivity, cancer predisposition).

3. Base Excision Repair (BER)

• Fixes small, non-bulky base lesions (e.g. cytosine → uracil via deamination).
• DNA glycosylase cleaves N-glycosidic bond → AP site.
• AP endonuclease nicks backbone; DNA Pol I/β insert correct base; ligase closes nick.

4. Double-Strand Break Repair

• Non-homologous End-Joining (NHEJ):
  • Ku70/Ku80, DNA-PKcs, ligase IV complex.
  • Error-prone (loss of nucleotides) → oncogenic translocations, immunodeficiency when defective.
• Homologous Recombination (HR):
  • Uses undamaged sister chromatid (late S/G₂ phase) as template → high fidelity.
  • BRCA1/2 pivotal; mutations elevate breast/ovarian cancer risk.

Clinical & Pharmacological Connections

• Fluoroquinolones (ciprofloxacin, levofloxacin) inhibit bacterial DNA gyrase → antibacterial therapy.
• Anti-cancer anthracyclines (doxorubicin) stabilise DNA-Topo II cleavage complex → apoptosis in rapidly dividing cells.
• PCNA over-expression as tumour proliferation index.
• Telomerase inhibitors & immunotherapies under investigation for malignancies.

Exam-Alert Summaries (memorise!)

• DNA synthesis direction: $5' \rightarrow 3'$; template read $3' \rightarrow 5'$.
• Leading vs. Lagging polymerases: Pol ε (leading), Pol δ (lagging) in eukaryotes.
• Sliding clamp names: β-clamp (prokaryotes) vs. PCNA (eukaryotes).
• Error-prone vs. high-fidelity DSB repair: NHEJ (error-prone); HR (error-free).
• Disease pairs:
  • Topo II ↔ doxorubicin cardiotoxicity monitoring.
  • NER defect ↔ xeroderma pigmentosum.
  • BRCA mutations ↔ HR deficiency ↔ PARP-inhibitor sensitivity.

Ethical, Philosophical & Practical Implications

• Manipulating replication & repair (e.g.
  CRISPR, telomerase modulation) raises questions on lifespan extension vs.
  carcinogenesis.
• Antibiotic resistance emerges when gyrase inhibitors are overused—public-health stewardship essential.
• Germ-line editing of repair pathways intersects with debates on genetic equity and “designer” traits.

Quick Reference – Numeric / Chemical Details

• Typical Okazaki fragment length (prokaryotes): $\approx 1000\text{–}2000\ \text{nt}$; eukaryotes: $\approx 100\text{–}200\ \text{nt}$.
• Mg²⁺ requirement for polymerase catalysis: $\approx 2\,\text{mM}$.
• Telomere repeat loss per division (human somatic): $50\text{–}200\ \text{bp}$.

Suggested Study & Practice

• Diagram replication fork labelling every protein; annotate directionality arrows.
• Work textbook end-of-chapter problems for BER vs. NER scenarios.
• Case studies: analyse patient with XP symptoms → map to NER gene defect.
• Simulate PCR primer design; rationalise need for $3'\text{-OH}$ similar to replication primer concept.