DNA Replication 1: Introduction & Prokaryotic Replication

🔁 dsDNA as a Template for Replication

• DNA is double-stranded and complementary, allowing it to act as its own template.

• This structure underlies its role in accurate replication.

🔬 Evidence for Semi-Conservative Replication

• Confirmed by the Meselson-Stahl experiment (not detailed here).

• Each daughter DNA molecule contains:

  • One original (parental) strand

  • One newly synthesised strand

🧪 DNA Polymerases

• Enzymes that synthesise DNA by adding deoxyribonucleotides (dNTPs) to a growing chain.

• Operate using a ssDNA template.

• All DNA polymerases:

  • Synthesise 5′→3′

  • Require a free 3′-OH group

  • Base-pair new dNTPs to the template strand

⚠ Why 5′→3′ only?

• 3′-OH attacks the α-phosphate of incoming dNTP.

• Forms a phosphodiester bond, releases pyrophosphate (PPi).

• Ensures continued chain elongation and error checking.

🔓 DNA Helicase (e.g., DnaB in bacteria)

• Unwinds dsDNA to give ssDNA templates.

• Powered by ATP hydrolysis.

• Combines with primase to form the primosome.

🧷 Replication Fork Structure

• DNA replication occurs at a replication fork.

• Leading strand: synthesised continuously in 5′→3′ direction.

• Lagging strand: synthesised discontinuously in Okazaki fragments.

🔹 Okazaki Fragments

• Short stretches of DNA (1,000–2,000 bp in prokaryotes)

• Joined later to form a continuous strand

• Occurs due to the antiparallel nature of DNA

🧬 DNA Polymerase III (Pol III) – Main Replicative Enzyme

• Major DNA polymerase in E. coli.

• Two main activities:

  • 5′→3′ DNA polymerase

  • 3′→5′ exonuclease (proofreading)

• Lacks 5′→3′ exonuclease (unlike pol I)

🔧 Structure:

• Core enzyme:

  • α (polymerase)

  • ε (proofreading exonuclease)

  • θ (stimulates ε)

• Works with β sliding clamp for processivity

🧲 Sliding Clamp & Clamp Loader

• β clamp: encircles DNA, keeps Pol III attached

• Clamp loader (γ complex): loads β clamp using ATP

🧫 DNA Primase (DnaG) – RNA Primer Formation

• RNA polymerase that synthesises short RNA primers (5–10 nt)

• Required because DNA polymerases cannot initiate synthesis

• Leading strand: 1 primer
  Lagging strand: new primer for every Okazaki fragment

🧹 Lagging Strand Maturation

Step-by-step process:

1. Pol III extends from RNA primer until next fragment.

2. DNA Pol I (polA):

   • Removes RNA primer (5′→3′ exonuclease)

   • Fills in gap with DNA

   • Proofreads (3′→5′ exonuclease)

3. DNA ligase seals the final nick between fragments.

🧷 Supporting Proteins

Single-Stranded Binding Proteins (SSBs)

• Bind ssDNA

• Prevent tangling or reannealing

• Stabilise template for replication

Topoisomerases

• Relieve tension ahead of fork due to unwinding

• Topo I: cuts one strand to relieve supercoils

• Topo II (DNA gyrase): cuts both strands to remove knots

🧪 The Prokaryotic Replication Fork – Complete Overview

• All components form a multi-enzyme complex.

• Ensures coordinated, high-fidelity replication.

🚀 Prokaryotic DNA Replication Initiation

oriC: Origin of Replication

• AT-rich region (~150–200 bp)

• Initiation is tightly regulated in E. coli.

Step-by-step:

1. DnaA binds oriC

   • Bends DNA, causes local unwinding at DUE (DNA Unwinding Element)

2. SSBs stabilise exposed ssDNA

3. DnaC loads DnaB helicase

4. DnaG primase recruited → forms primosome

↔ Bidirectional Replication

• Initiation leads to two replication forks moving away from oriC.

• Allows faster replication of circular chromosomes.

🛑 Termination in Prokaryotes

• ter sites located opposite oriC

• Each ter only halts forks from one direction

• Tus protein binds ter → blocks DnaB helicase

• Topoisomerases decatenate (unlink) chromosomes

🧠 Quick Summary