DNA REPLICATION, TRANSCRIPTION AND TRANSLATION Part-IV
Introduction
DNA replication is an extraordinarily accurate process. The human genome
contains approximately 3.2 billion base pairs, yet almost every daughter
cell receives an almost identical copy of DNA after each cell division.
This remarkable accuracy is achieved because cells possess:
- High-fidelity DNA polymerases
- Proofreading mechanisms
- DNA repair systems
- Cell-cycle checkpoints
Without these protective mechanisms, mutations would accumulate rapidly,
causing genetic diseases, developmental abnormalities, ageing, and cancer.
How Accurate Is DNA Replication?
· DNA polymerase alone
makes approximately: 1 error in 10⁵ nucleotides
· After proofreading: 1
error in 10⁷ nucleotides
· After DNA repair: Approximately
1 error in 10⁹–10¹⁰ nucleotides
This makes DNA replication one of the most accurate biological processes.
Why Is High Fidelity Necessary?
Accurate DNA replication ensures:
- Genetic stability
- Correct protein synthesis
- Normal growth
- Proper embryonic development
- Prevention of inherited disorders
- Prevention of cancer
Sources of DNA Damage
DNA is continuously damaged by both internal and external factors.
Endogenous (Internal)
- Reactive oxygen species (ROS)
- Replication errors
- Spontaneous base loss
(depurination)
- Spontaneous deamination
Exogenous (External)
- Ultraviolet (UV) radiation
- X-rays
- Gamma rays
- Chemical mutagens
- Environmental toxins
- Tobacco smoke
- Certain viruses
Proofreading
Definition
Proofreading is the immediate correction of wrongly inserted nucleotides
during DNA replication.
Enzyme Responsible- DNA polymerase
Many replicative DNA polymerases possess: 3' → 5' exonuclease activity
Mechanism
Step 1
Wrong nucleotide inserted.
↓
Step 2
DNA polymerase detects mismatch.
↓
Step 3
Incorrect nucleotide removed.
↓
Step 4
Correct nucleotide inserted.
↓
Replication continues.
Importance of Proofreading
- Prevents mutations
- Maintains genome stability
- Increases replication fidelity
- Ensures correct inheritance
DNA repair
Even after proofreading, some mistakes remain. Therefore, cells possess
specialized repair systems.
Types of DNA Repair
Major DNA repair mechanisms include:
- Mismatch Repair (MMR)
- Base Excision Repair (BER)
- Nucleotide Excision Repair (NER)
- Double-Strand Break Repair (DSBR)
1. Mismatch Repair (MMR)
Function
Corrects mismatched bases that escape proofreading.
Example- A paired with C, instead of- A paired with T
Steps
Mismatch detected
↓
Incorrect nucleotide removed
↓
Gap filled by DNA polymerase
↓
DNA ligase seals strand
Importance
Prevents permanent mutations after replication.
2. Base Excision Repair (BER)
Repairs
Small damaged bases such as:
- Oxidized bases
- Deaminated bases
- Alkylated bases
Steps
Damaged base recognized
↓
DNA glycosylase removes damaged base
↓
AP endonuclease cuts DNA backbone
↓
DNA polymerase inserts correct nucleotide
↓
DNA ligase seals nick
3. Nucleotide Excision Repair (NER)
Repairs- Large DNA lesions. Especially: UV-induced thymine dimers.
Mechanism
Damaged DNA segment removed
↓
DNA polymerase synthesizes replacement DNA
↓
DNA ligase joins strand
Clinical Importance
Defect causes: Xeroderma pigmentosum
Patients show:
- Extreme UV sensitivity
- Early skin cancers
- Defective nucleotide excision
repair
4. Double-Strand Break Repair
Double-strand DNA breaks are among the most dangerous forms of DNA
damage. Cells repair them mainly by:
Homologous Recombination (HR)
- Uses the sister chromatid as a
template.
- Highly accurate.
- Predominant in S and G₂ phases.
Non-Homologous End Joining (NHEJ)
- Directly joins broken DNA ends.
- Faster but more error-prone.
Cell-Cycle Checkpoints
Cells do not divide until DNA replication is complete and DNA damage is
repaired. Important checkpoints include:
G₁/S Checkpoint
- Verifies DNA integrity before
replication.
Intra-S Checkpoint
- Monitors ongoing DNA replication.
- Slows replication if damage is
detected.
G₂/M Checkpoint
- Ensures DNA replication is
complete.
- Prevents entry into mitosis if
DNA remains damaged.
Regulation Of DNA Replication
Replication occurs:
- Only once during each cell cycle.
- Only during the S phase.
This prevents over-replication or incomplete replication.
DNA Replication Inhibitors
Certain drugs inhibit DNA replication and are widely used in medicine.
|
Drug/Class |
Mechanism |
Clinical Use |
|
Quinolones (e.g., ciprofloxacin) |
Inhibit bacterial DNA gyrase
(Topoisomerase II) |
Bacterial infections |
|
Etoposide |
Inhibits Topoisomerase II |
Cancer chemotherapy |
|
Doxorubicin |
Interferes with topoisomerase and
DNA |
Cancer chemotherapy |
|
Acyclovir |
Inhibits viral DNA polymerase |
Herpes virus infections |
|
Zidovudine (AZT) |
Inhibits reverse transcriptase |
HIV infection |
Replication Errors and Mutations
If DNA damage is not repaired:
↓
Mutation occurs
↓
Altered protein
↓
Disease or altered phenotype
Some mutations are neutral, some harmful, and a few beneficial.
DNA Replication And Cancer
Cancer develops due to the accumulation of mutations in genes
controlling:
- Cell division
- DNA repair
- Apoptosis
Failure of proofreading and DNA repair greatly increases cancer risk.
Complete Flow Chart Of Dna Replication
Origin of Replication
│
▼
Helicase Unwinds DNA
│
▼
Topoisomerase Removes Supercoils
│
▼
SSB Proteins Stabilize DNA
│
▼
Primase Synthesizes RNA Primer
│
▼
DNA Polymerase Synthesizes New DNA
│
▼
Leading Strand ───────────────►
Continuous
Lagging Strand ───────────────►
Okazaki Fragments
│
▼
Primer Removal
│
▼
Gap Filling
│
▼
DNA Ligase Seals Nicks
│
▼
Proofreading
│
▼
DNA Repair (if required)
│
▼
Two Identical DNA Molecules
Complete Summary of DNA Replication
|
Feature |
Description |
|
Nature |
Semiconservative |
|
Direction |
5′ → 3′ synthesis |
|
Template read |
3′ → 5′ |
|
Occurs during |
S phase |
|
Replication type |
Bidirectional |
|
Leading strand |
Continuous |
|
Lagging strand |
Discontinuous |
|
Okazaki fragments |
Present only on lagging strand |
|
Main bacterial replicative enzyme |
DNA Polymerase III |
|
Primer removal (bacteria) |
DNA Polymerase I |
|
Primer removal (eukaryotes) |
RNase H and FEN1 |
|
Joining enzyme |
DNA Ligase |
|
Unwinding enzyme |
Helicase |
|
Supercoil removal |
Topoisomerase |
|
Primer synthesis |
Primase |
|
Fidelity |
~1 error per 10⁹–10¹⁰ nucleotides
after repair |
Important Scientists
|
Scientist |
Contribution |
|
James Watson |
Double-helix model |
|
Francis Crick |
Double-helix model and Central Dogma |
|
Matthew Meselson & Franklin
Stahl |
Proved semiconservative replication |
|
Arthur Kornberg |
Discovered DNA Polymerase I |
|
Reiji Okazaki |
Discovered Okazaki fragments |
|
J. Herbert Taylor |
Demonstrated semiconservative
replication in eukaryotes |
Memory Tricks for Exam
H-P-P-L Sequence
H → Helicase (opens
DNA)
↓
P → Primase (makes
primer)
↓
P → Polymerase
(extends DNA)
↓
L → Ligase (joins
fragments)
Leading vs Lagging
Leading = Long continuous
Lagging = Little pieces (Okazaki fragments)
Direction Rule
DNA Polymerase Always Adds to the 3′ End
Therefore,
DNA synthesis is always 5′ → 3′.
COMPLETE CHAPTER QUICK REVISION
- DNA replication is semiconservative.
- Occurs during the S phase.
- Begins at the origin of
replication.
- Helicase unwinds DNA.
- Topoisomerase relieves supercoiling.
- SSB proteins stabilize single strands.
- Primase synthesizes RNA primers.
- DNA Polymerase III synthesizes bacterial DNA.
- DNA is synthesized only in the 5′
→ 3′ direction.
- Leading strand is continuous.
- Lagging strand forms Okazaki fragments.
- DNA Polymerase I (bacteria) removes RNA primers.
- RNase H/FEN1 remove primers in eukaryotes.
- DNA Ligase seals nicks.
- Telomerase maintains chromosome ends.
- Proofreading and DNA repair
maintain high replication fidelity.
Summary
- DNA replication is
semiconservative, bidirectional, and template-directed.
- DNA polymerases require a primer
and synthesize DNA only in the 5′ → 3′ direction.
- Leading and lagging strands arise
because the two DNA strands are antiparallel.
- Okazaki fragments are synthesized
on the lagging strand and joined by DNA ligase.
- High fidelity of DNA replication
is achieved through complementary base pairing, proofreading, and DNA
repair mechanisms.