DNA REPLICATION, TRANSCRIPTION AND TRANSLATION Part-III

 

DNA REPLICATION, TRANSCRIPTION AND TRANSLATION Part-III

DNA Replication (Part 1C)

Mechanism of DNA Replication (Step-by-Step)

Introduction

After understanding the basic concepts of DNA replication (Part 1A) and the enzymes involved (Part 1B), we now study the actual mechanism of DNA replication.

DNA replication is one of the most accurate biological processes known. Millions of nucleotides are copied in the correct order within a short period with an error rate of only about 1 in 10⁹–10¹⁰ nucleotides after proofreading and repair.

The process consists of three major stages:

1. Initiation
2. Elongation
3. Termination

Overview Of DNA Replication

Origin of Replication

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DNA Unwinding (Helicase)

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RNA Primer Formation (Primase)

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DNA Synthesis (DNA Polymerase)

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Primer Removal

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Gap Filling

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DNA Ligase Seals Nicks

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Two Identical DNA Molecules

Step 1 — Initiation of DNA Replication

Initiation is the first stage of DNA replication. It begins at the Origin of Replication (Ori).

A. Recognition of Origin

Special initiator proteins recognize the origin sequence. In Escherichia coli, these proteins bind to OriC. This marks the site where replication begins.

B. DNA Unwinding

Enzyme: DNA Helicase

Helicase:

• Breaks hydrogen bonds.
• Separates the two DNA strands.
• Opens the double helix.

Result: Formation of a Replication Bubble.

Before

 

====================

 

After

 

======<      >======

C. Formation of Replication Forks

Each replication bubble possesses two replication forks. Replication proceeds simultaneously in opposite directions. Therefore, DNA replication is Bidirectional.

D. Stabilization of Single Strands

Protein Involved

Single-Strand Binding Proteins (SSBs)

Functions:

• Prevent DNA strands from rejoining.
• Protect DNA.
• Stabilize replication fork.

E. Removal of Supercoiling

Helicase creates torsional strain.

Enzyme

Topoisomerase

In bacteria: DNA Gyrase

Function: Removes supercoils ahead of replication fork.

Step 2 — Primer Synthesis

DNA polymerase cannot start DNA synthesis.

Therefore, RNA primers are synthesized.

Enzyme- Primase

Primer: Short RNA segment

Provides: Free 3'-OH group, Necessary for DNA polymerase.

Step 3 — DNA Chain Elongation

Main Enzyme- DNA Polymerase

IMPORTANT RULE

DNA polymerase always synthesizes DNA

5' → 3'

while reading the template

3' → 5'

Why Are Two Strands Synthesized Differently?

The two parental DNA strands are antiparallel.

Therefore, One strand can be copied continuously.

The other cannot. Hence- Two different modes of synthesis occur.

Leading Strand

Definition

The strand synthesized continuously toward the replication fork.

Characteristics:

• Continuous synthesis
• Only one RNA primer needed
• Faster synthesis

Diagram

Replication Fork →

 

Template

 

3' -----------------5'

 

↓

 

Continuous DNA synthesis

 

5' -----------------3'

Lagging Strand

Definition

The strand synthesized discontinuously away from the replication fork.

Characteristics:

• Discontinuous synthesis
• Many RNA primers required
• Slower synthesis

Diagram

Replication Fork →

 

5' ----------------3'

 

↓

 

Short DNA pieces

 

□□□□ □□□□ □□□□

These short pieces are called Okazaki Fragments.

Okazaki Fragments

Definition

Short DNA fragments synthesized on the lagging strand during replication.

Discovered by: Reiji Okazaki and Tsuneko Okazaki.

Approximate Length

Prokaryotes- 1000–2000 nucleotides

Eukaryotes- 100–200 nucleotides

Why Are Okazaki Fragments Formed?

DNA polymerase works only

5' → 3'

Since the lagging strand runs in the opposite orientation, DNA must be synthesized in small fragments.

DNA Synthesis on Both Strands

Leading Strand	Lagging Strand
Continuous	Discontinuous
One primer	Many primers
No Okazaki fragments	Okazaki fragments present
Faster	Slightly slower

Step 4 — Removal of RNA Primers

RNA primers are temporary. They must be removed.

In Prokaryotes

DNA Polymerase I removes primers. It replaces RNA with DNA.

In Eukaryotes

RNA primers are removed by:

• RNase H
• Flap Endonuclease (FEN1)

DNA Polymerase fills the resulting gaps.

Step 5 — Ligation

After primer removal, DNA fragments remain separated.

Enzyme

DNA Ligase

Function

Forms phosphodiester bonds. Joins Okazaki fragments. Produces a continuous DNA strand.

Step 6 — Termination

Replication continues until: Entire chromosome is copied.

In Prokaryotes

Replication forks meet at termination (Ter) sites. Replication stops. Two daughter DNA molecules separate.

In Eukaryotes

Replication forks from adjacent origins eventually meet and fuse. Replication terminates after the entire chromosome has been duplicated.

Why Is DNA Replication Called Semi-Discontinuous?

One strand

↓

Continuous

Other strand

↓

Discontinuous

Therefore,

Replication is called Semi-discontinuous.

Why Is DNA Replication Bidirectional?

Replication proceeds simultaneously in opposite directions from each origin. Thus, Two replication forks move away from the origin.

Why Is DNA Replication Highly Accurate?

Several factors ensure accuracy.

1. Complementary Base Pairing

A pairs with T

G pairs with C

2. Proofreading

DNA polymerases possess 3' → 5' exonuclease activity. Wrong nucleotide removed immediately.

3. DNA Repair Mechanisms

Remaining mistakes corrected after replication.

DNA Replication Speed

Prokaryotes

Approximately 1000 nucleotides/second

Eukaryotes

Approximately 50 nucleotides/second, although slower, eukaryotes compensate by using multiple origins of replication.

Replication Of Circular DNA

Occurs in bacteria. Replication proceeds around the circular chromosome until completion.

Replication Of Linear DNA

Occurs in eukaryotes. Special problem arises at chromosome ends. This is called the End Replication Problem.

The End Replication Problem

After removal of the final RNA primer on the lagging strand, there is no upstream 3′-OH group available for DNA polymerase to fill the gap.

As a result, the newly synthesized lagging strand becomes slightly shorter after each round of replication. If uncorrected, chromosomes would progressively shorten with every cell division.

Telomeres

Definition

Telomeres are repetitive, non-coding DNA sequences present at the ends of linear eukaryotic chromosomes.

In humans, the repeat sequence is: TTAGGG, repeated thousands of times.

Functions Of Telomeres

• Protect chromosome ends from degradation.
• Prevent chromosome ends from fusing with one another.
• Maintain chromosome stability.
• Buffer the loss of DNA during replication.

Telomerase

Definition

Telomerase is a specialized ribonucleoprotein enzyme that extends telomeres by adding repetitive DNA sequences to chromosome ends.

It contains:

• A protein component with reverse transcriptase activity.
• An intrinsic RNA molecule that serves as the template.

Where Is Telomerase Active?

High activity in:

• Germ cells
• Stem cells
• Early embryonic cells

Low or absent activity in:

• Most adult somatic cells

Many cancer cells reactivate telomerase, allowing unlimited cell division.

Significance Of Telomerase

• Prevents excessive chromosome shortening.
• Maintains genomic integrity.
• Contributes to cellular longevity in specific cell types.

Flow Chart of DNA Replication

Origin of Replication

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Helicase Unwinds DNA

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Replication Fork Forms

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Primase Synthesizes RNA Primers

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DNA Polymerase Extends DNA

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├───────────────┐

▼                                          ▼

Leading Strand      Lagging Strand

Continuous          Okazaki Fragments

    │                                           │

    └──────┬────────┘

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Primer Removal

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Gap Filling

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DNA Ligase Seals Nicks

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Termination

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Two Identical DNA Molecules

Leading Vs Lagging Strand

Feature	Leading Strand	Lagging Strand
Direction of synthesis	Toward replication fork	Away from replication fork
Nature	Continuous	Discontinuous
RNA primers	One	Many
Okazaki fragments	Absent	Present
DNA ligase requirement	Minimal	Essential

 

Prokaryotic vs Eukaryotic DNA Replication

Feature	Prokaryotes	Eukaryotes
Chromosomes	Circular	Linear
Origins of replication	Single	Multiple
Replication speed	~1000 nucleotides/s	~50 nucleotides/s
Okazaki fragment size	1000–2000 nt	100–200 nt
Main replicative polymerase	DNA Polymerase III	DNA Polymerases δ and ε
Primer removal	DNA Polymerase I	RNase H and FEN1
Telomeres	Absent	Present
Telomerase	Not required	Required for telomere maintenance

Clinical Correlation

DNA Repair Defects

Inherited defects in DNA repair pathways can lead to genomic instability and increased cancer risk.

Example: Xeroderma pigmentosum is caused by defective nucleotide excision repair, resulting in extreme sensitivity to ultraviolet (UV) radiation.

High-Yield Facts

• DNA polymerases can only extend a pre-existing primer and cannot initiate DNA synthesis.
• The antiparallel arrangement of DNA strands results in continuous synthesis on the leading strand and discontinuous synthesis on the lagging strand.
• Okazaki fragments are formed only on the lagging strand and are subsequently joined by DNA ligase.
• DNA replication is bidirectional, semiconservative, and semi-discontinuous.
• Multiple origins of replication in eukaryotic chromosomes enable timely duplication of the large genome during the S phase.