Learning Objective: To explain the molecular steps of DNA replication—covering initiation, elongation, enzymatic functions, proofreading, telomere maintenance, and pharmacologic inhibitors—so learners can describe how accurate and complete duplication of the genome occurs in prokaryotic and eukaryotic cells.

DNA replication is a semiconservative, bidirectional process in which each daughter DNA molecule receives one parental and one newly synthesized strand. Although the overall process is similar in prokaryotes and eukaryotes, key enzymes and regulatory mechanisms differ.

Key Steps of DNA Replication

1. Recognition of the Origin

Replication begins when specific proteins recognize the origin of replication (Ori).

Prokaryotes: single origin (OriC)
Eukaryotes: multiple origins per chromosome

2. Helicase-Mediated Unwinding

Helicase breaks hydrogen bonds between complementary bases, separating the two parental strands.
This form:

Two replication forks
A replication bubble

3. Stabilization of Single-Stranded DNA

The unwound DNA is unstable. Single-Stranded DNA Binding Proteins (SSB):

Prevent reannealing of complementary strands
Protect ssDNA from nuclease digestion

4. Primer Synthesis

Primase (an RNA polymerase):

Synthesizes a short RNA primer (~10 nt)
Provides a free 3′-OH for DNA polymerases (they cannot initiate synthesis)

5. DNA Polymerase III / δ-driven Extension

Synthesis always occurs 5′ → 3′.

Leading Strand

Synthesized continuously toward the replication fork
Requires one primer

Lagging Strand

Synthesized discontinuously away from the fork
Forms Okazaki fragments
Each fragment requires a new primer

There is a leading and lagging strand at each fork, resulting in two of each per origin.

6. Removal of RNA Primers and Replacement

Eukaryotes

RNAse H removes RNA primers
A DNA polymerase fills the gap with DNA

Prokaryotes

DNA polymerase I
- Removes RNA primers (5′ → 3′ exonuclease activity)
- Fills in with DNA

7. Proofreading

Both prokaryotic and eukaryotic polymerases possess 3′ → 5′ exonuclease proofreading.
If an incorrect base is inserted:

The enzyme removes the mismatched nucleotide
Synthesis resumes accurately

8. DNA Ligase Seals the Nicks

DNA ligase forms the final phosphodiester bonds between Okazaki fragments, creating a continuous DNA strand.

9. DNA Gyrase / Topoisomerase II Action

As helicase unwinds DNA, positive supercoils accumulate ahead of the fork.

DNA Gyrase (Prokaryotic Topoisomerase II):

Creates transient double-strand breaks
Introduces negative supercoils
Relieves torsional stress

Drugs:

Quinolones / Fluoroquinolones (e.g., ciprofloxacin, levofloxacin)
- Inhibit DNA gyrase
- Block replication → bactericidal
Eukaryotic Topoisomerase II inhibitors:
- Etoposide, teniposide (anticancer)

Eukaryotic DNA Polymerases

Polymerase	Function
α	Initiator; works with primase; begins DNA synthesis
δ	The main enzyme for the lagging strand, also for the leading strand
ε	DNA repair can substitute for δ
β	Base excision repair
γ	Replicates mitochondrial DNA