4.1 Introduction
DNA replication is a
highly regulated, semi-conservative process that ensures the accurate
duplication of the genetic material in all living cells. It is essential for
cell division and is a cornerstone of genetic inheritance. This chapter
discusses the molecular mechanisms, enzymatic players, initiation, elongation,
termination, and regulation of DNA replication in prokaryotes and eukaryotes.
4.2 The Semi-Conservative Model of
Replication
Proposed by Watson and
Crick and experimentally confirmed by the Meselson-Stahl experiment, the
semi-conservative model states that each daughter DNA molecule contains one
original (parental) strand and one newly synthesized strand.
4.3 Basic Requirements for DNA Replication
- Template
DNA: Provides the sequence to be copied.
- Primers:
Short RNA segments required to start DNA synthesis.
- DNA
polymerase: Enzyme that catalyzes the addition
of nucleotides.
- dNTPs:
Deoxyribonucleoside triphosphates (dATP, dTTP, dGTP, dCTP) serve as
building blocks.
- Replication
enzymes and proteins: Helicase, primase, ligase,
topoisomerase, and single-strand binding proteins (SSBs).
4.4 Key Enzymes and Proteins in DNA
Replication
|
Enzyme/Protein |
Function |
|
DNA Helicase |
Unwinds the double helix at the
replication fork. |
|
Single-Strand Binding Proteins (SSBs) |
Prevent reannealing of separated
strands. |
|
Topoisomerase |
Relieves supercoiling ahead of the
replication fork. |
|
Primase |
Synthesizes RNA primers to initiate
synthesis. |
|
DNA Polymerase |
Adds nucleotides to the growing DNA
chain. |
|
DNA Ligase |
Seals the nicks between Okazaki
fragments. |
4.5 Directionality and the Replication
Fork
DNA polymerases can only synthesize DNA in the 5'
to 3' direction. This leads to the formation of:
- Leading
strand: Synthesized continuously toward the
replication fork.
- Lagging strand: Synthesized discontinuously as Okazaki fragments away from the fork.
4.6 Steps in DNA Replication
4.6.1 Initiation
Prokaryotes:
- Begins
at a single origin of replication (OriC in E. coli).
- DnaA
proteins bind to OriC and recruit helicase (DnaB).
- Primase
synthesizes RNA primers.
Eukaryotes:
- Multiple
origins of replication.
- Origin
Recognition Complex (ORC) binds to origins.
- Licensing
factors ensure each origin is used once per cycle.
4.6.2 Elongation
- DNA
polymerase III (in prokaryotes) adds nucleotides to the 3' end of the
primer.
- The
leading strand is synthesized continuously.
- The
lagging strand forms Okazaki fragments.
- DNA
polymerase I replaces RNA primers with DNA (in prokaryotes).
- DNA
ligase joins Okazaki fragments.
4.6.3 Termination
Prokaryotes:
- Termination
sequences (Ter sites) bind Tus proteins to halt replication.
- Circular
chromosomes are resolved by topoisomerase.
Eukaryotes:
- Linear
chromosomes pose end-replication problems.
- Telomerase
adds repetitive sequences to telomeres to prevent shortening.
4.7 DNA Polymerases in Different Organisms
Prokaryotic DNA Polymerases:
- DNA
Pol I: Removes RNA primers and fills in
DNA.
- DNA
Pol II: Involved in repair.
- DNA
Pol III: Major replication enzyme.
Eukaryotic DNA Polymerases:
- Pol
α: Initiates replication with primase activity.
- Pol
δ: Extends lagging strand.
- Pol
ε: Extends leading strand.
- Pol β, γ, η: Involved in repair and mitochondrial replication.
4.8 Regulation of DNA Replication
- Cell
Cycle Control: Initiation is tightly regulated by
cyclin-dependent kinases (CDKs).
- Replication
Licensing: Prevents re-replication via proteins
like geminin and CDT1.
- Checkpoint Proteins: ATR and ATM kinases monitor DNA integrity during replication.
4.9 Replication in Special Cases
4.9.1 Telomere Replication
- Telomerase
extends the 3' end of linear chromosomes.
- Important
in germ cells, stem cells, and cancer cells.
4.9.2 Mitochondrial DNA Replication
- Occurs
separately from nuclear DNA replication.
- Involves
DNA polymerase γ.
- Autoradiography:
Detects newly synthesized DNA using labeled nucleotides.
- Density
Gradient Centrifugation: Used in the
Meselson-Stahl experiment.
- Pulse-Chase
Experiments: Analyze the timing and pattern of
DNA synthesis.
- DNA Fiber Assays: Visualize replication fork movement.
4.11 Clinical and Biotechnological
Implications
- Cancer
Biology: Aberrant replication can lead to
genomic instability.
- Antibiotics:
Some drugs target bacterial replication enzymes (e.g., quinolones target
topoisomerase).
- PCR
Technique: Mimics replication in vitro using
thermostable DNA polymerase.
- Gene Editing: Techniques like CRISPR rely on cellular DNA repair and replication mechanisms.
1. Which of the following enzymes is
responsible for removing RNA primers in prokaryotic DNA replication?
A) DNA polymerase III
B) DNA polymerase I
C) Helicase
D) DNA ligase
Answer: B) DNA polymerase
I
Explanation: DNA Pol I removes RNA primers using its 5’→3’ exonuclease
activity and replaces them with DNA.
2. What is the function of helicase during
DNA replication?
A) Adds nucleotides to the growing DNA chain
B) Seals nicks between Okazaki fragments
C) Breaks hydrogen bonds between DNA strands
D) Synthesizes RNA primers
Answer: C) Breaks
hydrogen bonds between DNA strands
Explanation: Helicase unwinds the DNA double helix by breaking hydrogen
bonds at the replication fork.
3. In eukaryotic cells, which enzyme is
primarily responsible for synthesis of the lagging strand?
A) DNA polymerase ε
B) DNA polymerase α
C) DNA polymerase δ
D) DNA polymerase β
Answer: C) DNA polymerase
δ
Explanation: Pol δ synthesizes the lagging strand in eukaryotes, while
Pol ε synthesizes the leading strand.
4. Okazaki fragments are joined together
by:
A) DNA polymerase I
B) Primase
C) DNA ligase
D) Helicase
Answer: C) DNA ligase
Explanation: DNA ligase forms phosphodiester bonds to seal gaps between
Okazaki fragments.
5. What is the role of single-strand
binding proteins (SSBs)?
A) Join DNA fragments
B) Unwind DNA helix
C) Stabilize unwound DNA strands
D) Prevent telomere shortening
Answer: C) Stabilize
unwound DNA strands
Explanation: SSBs prevent the reannealing of DNA strands after they are
separated by helicase.
6. Which statement about DNA polymerase is
false?
A) It synthesizes DNA in 5’ to 3’ direction.
B) It requires a primer to initiate synthesis.
C) It can add nucleotides without a template.
D) It has proofreading ability.
Answer: C) It can add
nucleotides without a template.
Explanation: DNA polymerase needs both a template strand and a primer to
begin DNA synthesis.
7. Telomerase is highly active in which
type of cells?
A) Neurons
B) Muscle cells
C) Somatic cells
D) Cancer cells
Answer: D) Cancer cells
Explanation: Telomerase is reactivated in many cancer cells, helping
them divide indefinitely.
8. In the Meselson-Stahl experiment, what
technique was used to separate DNA strands based on density?
A) Gel electrophoresis
B) Autoradiography
C) Density gradient centrifugation
D) Chromatography
Answer: C) Density
gradient centrifugation
Explanation: Meselson and Stahl used cesium chloride (CsCl) gradient
centrifugation to prove semi-conservative replication.
9. Which DNA replication enzyme has both
5'→3' polymerase and 3'→5' exonuclease activity in prokaryotes?
A) DNA polymerase III
B) DNA polymerase I
C) Primase
D) Ligase
Answer: A) DNA polymerase
III
Explanation: Pol III adds nucleotides (5'→3') and also proofreads using
its 3'→5' exonuclease function.
10. In which phase of the eukaryotic cell
cycle does DNA replication occur?
A) G1 phase
B) S phase
C) G2 phase
D) M phase
Answer: B) S phase
Explanation: DNA replication takes place during the Synthesis (S) phase
of the cell cycle.
4.13 References
1.
Alberts, B., Johnson, A., Lewis, J., et
al. (2015). Molecular Biology of the Cell (6th ed.). Garland Science.
2.
Watson, J. D., et al. (2013). Molecular
Biology of the Gene (7th ed.). Pearson.
3.
Kornberg, A., & Baker, T. A. (2005). DNA
Replication (2nd ed.). University Science Books.
4.
Meselson, M., & Stahl, F. W. (1958).
The replication of DNA in Escherichia coli. PNAS, 44(7), 671–682.
5.
Bell, S. P., & Dutta, A. (2002). DNA
replication in eukaryotic cells. Annual Review of Biochemistry, 71,
333–374.
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