DNA REPLICATION: MECHANISM & ENZYMES

[Ch-05: Molecular Basis of Inheritance]

Summary of Content:

1. Key Enzymes & Proteins Table:

o Helicase: Unwinding double helix at the fork.
o Topoisomerase / DNA Gyrase: Relieving supercoiling & strain.
o Primase: Synthesizing RNA primers for 3'-OH extension.
o DNA Polymerase III: Primary 5'  3' elongation enzyme.
o DNA Polymerase I: Primer removal (5' 3' exonuclease) and gap filling.
o DNA Ligase: Joining Okazaki fragments via phosphodiester bonds.
o SSBPs: Single-strand stabilization against re-annealing.
2. Mechanism of DNA Replication (Step-by-Step):
o Initiation: Ori site selection, unwinding, and replication fork assembly.
o Primer Synthesis: RNA priming requirements.
o Elongation: Detailed breakdown of continuous (leading strand) vs. discontinuous
(lagging strand / Okazaki fragments) synthesis.
o Termination & Ligation: Primer degradation, DNA repair filling, and
phosphodiester backbone sealing.
o Key Assessment Callout: Explanation of why synthesis occurs exclusively in the
5' 3' direction.

Key Enzymes & Proteins Involved in DNA Replication

Enzyme / Factor Key Function & Role in Replication

Helicase Unwinds the double helix by breaking hydrogen bonds

between complementary nitrogenous base pairs, creating the
replication fork.

Topoisomerase / DNA Relieves supercoiling and torsional strain ahead of the

Gyrase advancing replication fork by cutting and resealing DNA
backbones.

Primase (RNA Polymerase) Synthesizes a short RNA primer (~10–20 nucleotides) to

provide a free 3'-OH end required for DNA polymerase
activation.

DNA Polymerase III Main replicating enzyme; adds deoxyribonucleotides

(dNTPs) exclusively in the 5' → 3' direction with high

Ch-05: Molecular Basis of Inheritance DNA Replication By Dr. Anita Subudhi

 fidelity.

DNA Polymerase I Removes RNA primers using 5' → 3' exonuclease activity
and replaces them with corresponding DNA nucleotides.

DNA Ligase Joins Okazaki fragments together on the lagging strand by

catalyzing phosphodiester bond formation ('molecular glue').

Single-Stranded Binding Binds to single-stranded template DNA to stabilize open

Proteins (SSBPs) strands and prevent premature re-annealing.

Step-by-Step Mechanism of DNA Replication

DNA replication is a semiconservative process occurring during the S phase (Synthesis phase)
of the cell cycle, ensuring exact duplication of the genome prior to cell division.

Step 1: Initiation & Unwinding at Origin of Replication

Replication begins at specific genomic loci termed the Origin of Replication (ori). Helicase
unwinds the double-stranded DNA by disrupting hydrogen bonds between base pairs,
generating a Y-shaped replication fork. Single-Stranded Binding Proteins (SSBPs) attach to
exposed strands to prevent re-annealing, while Topoisomerase (DNA Gyrase) relieves
supercoiling tension accumulated ahead of the fork.

Structure of the DNA Replication Fork

Ch-05: Molecular Basis of Inheritance DNA Replication By Dr. Anita Subudhi

Step 2: Primer Synthesis
DNA polymerases cannot initiate polynucleotide synthesis de novo because they require an
existing 3'-OH group to attach incoming nucleotides. Primase synthesizes a short RNA primer
(~10-20 nucleotides) complementary to the template strand, creating the necessary starter
block.

Step 3: Elongation (Continuous vs. Discontinuous Synthesis)

DNA Polymerase III adds deoxyribonucleotides (dNTPs) exclusively in the 5' → 3' direction:
• Leading Strand: Synthesized continuously toward the replication fork using the 3' → 5'
template strand. Requires only a single RNA primer.
• Lagging Strand: Synthesized discontinuously away from the replication fork using the 5'
→ 3' template strand, producing short stretches known as Okazaki fragments. Each fragment
requires its own RNA primer.

Step 4: Primer Removal, Gap Filling & Ligation

DNA Polymerase I removes RNA primers via its 5' → 3' exonuclease activity and fills the
resulting gaps with complementary DNA nucleotides. Finally, DNA Ligase catalyzes
phosphodiester bonds between neighboring Okazaki fragments, sealing nicks in the sugar-
phosphate backbone.

Key Assessment Concept: Unidirectional Polymerization

Ch-05: Molecular Basis of Inheritance DNA Replication By Dr. Anita Subudhi

 DNA polymerases can only catalyze polymerization in the 5' → 3' direction. This restriction
occurs because the enzyme requires a free 3'-OH group to attach the incoming
deoxyribonucleoside triphosphate (dNTP), leading to the antiparallel continuous (leading)
and discontinuous (lagging) synthesis at the replication fork.

Features of Eukaryotic DNA Replication

 Replication is bi-directional and originates at multiple origins of replication (Ori C)
in eukaryotes.
 DNA replication uses a semi-conservative method that results in a double-stranded
DNA with one parental strand and a new daughter strand.
 It occurs only in the S phase and at many chromosomal origins.
 Takes place in the cell nucleus.
 Synthesis occurs only in the 5′to 3′direction.
 Individual strands of DNA are manufactured in different directions, producing a
leading and a lagging strand.
 Lagging strands are created by the production of small DNA fragments called
Okazaki fragments that are eventually joined together.
 Eukaryotic cells possess five types of polymerases involved in the replication
process.
DNA Polymerases:
 Eukaryotic cells contain five different DNA polymerases; α, β, γ, δ and ε.
 DNA polymerases α and δ replicate chromosomal DNA, DNA polymerases β and ε
repair DNA, and DNA polymerase γ replicates mitochondrial DNA.
 DNA polymerase α and δ synthesize the lagging strand, via Okazaki fragments.
 The RNA primers are synthesized by DNA polymerase α which carries a
primase subunit.
 DNA polymerase ε synthesizes the leading strand.
 Telomerase, a DNA polymerase that contains an integral RNA that acts as its own
primer, is used to replicate DNA at the ends of chromosomes (telomeres).
 DNA topoisomerase I: Relaxes the DNA helix during replication through creation
of a nick in one of the DNA strands.
 DNA topoisomerase II: Relieves the strain on the DNA helix during replication by
forming supercoils in the helix through the creation of nicks in both strands of DNA.
 DNA ligase: Forms a 3′-5′phosphodiester bond between adjacent fragments of
DNA.
Process of Eukaryotic DNA Replication
 Replication of each linear DNA molecule in a chromosome starts at many origins,
one every 30–300 kb of DNA depending on the species and tissue, and proceeds bi-
directionally from each origin.

Ch-05: Molecular Basis of Inheritance DNA Replication By Dr. Anita Subudhi

  At each origin, a replication bubble forms consisting of two replication forks moving
in opposite directions. The DNA replicated under the control of a single origin is
called a replicon. DNA synthesis proceeds until replication bubbles merge together.
 At the origin, enzymes unwind the double helix making its components accessible
for replication.
 The helix is unwound by helicase to form a pair of replication forks.
 The unwound helix is stabilized by SSB proteins and DNA topoisomerases.
 The RNA primers required are made by DNA polymerase α which carries a primase
subunit.
 DNA polymerase α initiates synthesis of the lagging strand, making first the RNA
primer and then extending it with a short region of DNA.
 DNA polymerase δ then synthesizes the rest of the Okazaki fragment.
 The leading strand is synthesized by DNA polymerase ε.
 The leading strand is synthesized continuously in the 5′to 3′ direction while the
lagging strand is synthesized discontinuously in the 5′to 3′ direction through the
formation of Okazaki fragments.
 At the completion of synthesis, DNA ligase seals the breaks between the Okazaki
fragments as well as around the primers to form continuous strands.
DNA Proofreading
In eukaryotes only the polymerases that deal with the elongation (delta and epsilon)
have proofreading ability (3’ → 5’ exonuclease activity).
If an error is detected, the erroneous base is removed via 3′to 5′exonuclease activity
replaced with the correct base.
Excision repair:
Removes pyrimidine dimers formed by UV rays or other mutated bases and replaces
them.
Significance of Eukaryotic DNA Replication
 DNA replication is a fundamental genetic process that is essential for cell growth
and division.
 DNA replication involve the generation of a new molecule of nucleic acid, DNA,
crucial for life.
 DNA replication is important for properly regulating the growth and division of
cells.
 It conserves the entire genome for the next generation.

Ch-05: Molecular Basis of Inheritance DNA Replication By Dr. Anita Subudhi