DNA replication

Replication Essentials - The Ground Rules

• Semiconservative: Each new DNA molecule contains one parent (old) strand and one daughter (new) strand.
• Origin of Replication (ORI):
  • Starting point; typically AT-rich (fewer H-bonds).
  • Prokaryotes have a single ORI; Eukaryotes have multiple ORIs.
• Bidirectional: Replication proceeds in both directions from the ORI, forming two replication forks.

• 5' → 3' Synthesis: DNA polymerase adds nucleotides ONLY to the free 3'-hydroxyl end of a growing strand.

⭐ The 5' → 3' directionality of DNA polymerase is absolute and dictates the existence of a continuous leading strand and a discontinuous lagging strand (Okazaki fragments).

The Replication Fork - Unzipping Action

• Helicase: Unzips the DNA double helix at the replication fork, breaking hydrogen bonds in an ATP-dependent process.
• Single-Strand Binding Proteins (SSBPs): Bind to the separated strands to keep them from re-annealing and protect them from nuclease degradation.
• DNA Topoisomerases: Relieve the torsional strain and negative supercoils that build up ahead of the fork.
  • Type I: Creates single-strand nicks to relax supercoils.
  • Type II (DNA Gyrase in E. coli): Induces double-strand breaks to manage supercoils.

⭐ Fluoroquinolones inhibit prokaryotic Type II topoisomerase (DNA gyrase). Etoposide/teniposide inhibit human Type II topoisomerase.

Prokaryote vs Eukaryote - Tale of Two Cells

• Origin of Replication: Single in prokaryotes vs. multiple in eukaryotes, allowing for faster overall replication despite a slower polymerase speed.
• DNA Polymerases:
  • Prokaryotes: Pol I, II, III. 📌 Pol III is the primary replicative enzyme.
  • Eukaryotes: Complex (α, δ, ε).
• Telomerase: Absent in prokaryotes (circular DNA); present in eukaryotes to maintain telomere length on linear chromosomes.
• Okazaki Fragments: Longer in prokaryotes; shorter in eukaryotes.

⭐ Eukaryotic DNA Pol α has primase activity and initiates synthesis. Pol δ synthesizes the lagging strand, and Pol ε synthesizes the leading strand.

Telomeres & Drugs - Ends and Blockers

• Telomeres: Repetitive DNA sequences (TTAGGG) at chromosome ends protecting against degradation. They shorten with each cell division, contributing to aging.
• Telomerase: A reverse transcriptase that lengthens telomeres.
  • High activity in stem, germ, and cancer cells.
  • Uses an internal RNA template to add repeats.
• Replication Inhibitors:
  • Hydroxyurea: Blocks ribonucleotide reductase, inhibiting dNTP synthesis.
  • 5-FU, Methotrexate: Inhibit thymidylate synthesis.

⭐ Telomerase reactivation is a hallmark of ~90% of human cancers, allowing them to evade senescence and achieve cellular "immortality."

High‑Yield Points - ⚡ Biggest Takeaways

• DNA replication is semiconservative and always proceeds in the 5' to 3' direction.
• Helicase unwinds the DNA helix, while topoisomerases (e.g., DNA gyrase) relieve supercoiling.
• Primase synthesizes an RNA primer to initiate DNA synthesis.
• DNA Polymerase III is the primary enzyme for synthesizing new DNA in prokaryotes.
• The lagging strand is synthesized discontinuously, creating Okazaki fragments.
• DNA Polymerase I removes RNA primers, and DNA ligase joins the fragments.