REPLICATION

Semi-conservative replication of Chromosomes in eukaryotes:

Autoradiography experiment in Vicia faba, by J.H. Taylor and his co-workers for the study of duplicating chromosomes in the root tip cells were first published in 1957.
They reported that DNA in all the organisms has the inherent capacity of self-replication.
The mechanism of DNA replication is so precise that all the cells derived from a zygote contain exactly similar DNA both in terms of quality and quantity.
The replication takes place in interphase after every cell division.

Theoretically, there may be following three possible modes of DNA replication:

Dispersive Method
Conservative Method
Semiconservative Method

Semiconservative mode is the most accepted of all.

DNA Replication is Semi-Conservative:

Watson and Crick model suggested that DNA replication is semi-conservative.
It implies that half of the DNA is conserved.
Only one new strand is synthesized, the other strand is the original DNA strand (template) that is retained.
Each parental DNA strand serves as a template for one new complementary strand.
The new strand is hydrogen bonded to its parental template strand and forms a double helix.
Each of these strands of the double helix contains one original parental strand and one newly formed strand.

Meselson and Stahl Experiment:

Mathew Meselson and Franklin Stahl proved experimentally that parental strands of a helix are distributed equally between the two daughter molecules.
They made use of the heavy isotope ¹⁵N as a tag to differentially label the parental strands. E. coli was grown in a medium containing ¹⁵N labeled NH₄Cl.

In this way both strands of DNA molecules were labeled with radioactive heavy isotope ¹⁵N in their purines and pyrimidines.
Therefore both strands were heavy or H DNA.
The bacteria were then transferred into a medium containing the common non-radioactive nitrogen ¹⁴N, which is a light medium.
It was found that after one cell division daughter molecules had one ¹⁵N strand the other ¹⁴N strand.
So this is a hybrid molecule, a heavy light of HL.

Fig. Semiconservative replication.

STEPS OF DNA REPLICATION

DNA-Replication

Stages of Replication

The DNA synthesis or replication can be divided into three stages

(1) Initiation

(1) Elongation

(3) Termination

(i) Initiation :

In E coli replication is origin a specific site which is called ori C.
It is highly conserved sequence which is 245 bp long. .
In this sequences there are three repeats of 13 bp AT rich sequence and four repeats of a 9 bp sequence.

At least, nine different enzymes/ proteins participate in the initiation phase
A single complex of four to five Dna A protein molecules binds to the four 9 bp repeats in the ori C
DnaA protein forms a complex of 30-40 molecules, each bound to an ATP molecule and bacterial histone like proteins around which the OriC DNA becomes wrapped
This process causes negatively supercoiling in DNA.
This melting (unwoundıng) of three 13 bp AT-rich repeat sequences.
After this Dna B with the help of Dna C protein loads on DNA.
It is a hexamer ring structure with two rings.
The two ring of Dna B, and DNA C complex loaded on to each DNA strand and unwinds the DNA bidirectionally by using energy of ATP hydrolysis to move into and melt double stranded DNA
The single stranded bubble created in this way is coated with single strand binding protein (SSB) to protect it from breakage and prevent DNA renaturation.
The enzyme DNA primase then attaches to the DNA and Synthesizes a short RNA primer to initiate synthesis of the leading stand of the first replication fork
Bidirectional replication then follows

Unwinding :

In replication DNA helicases travel along the template strands to open the double helix for copying the nucleotide on DNA template
In addition to Dna B. a second DNA helicase may bind to the other strand to assist unwinding
In a closed circular DNA molecule, removal of helical tums at the replication forks leads to the introduction of additional tums in the rest of the molecule in the form of positive supercoiling
Although the natural negative supercoiling of circular DNA partially compensates for this, it is insufficient to allow continued progression of the replication forks
This positive supercoiling must be relaxed continuously by the introduction of further negative supercoils by type Il topoisomerases (eg DNA gyrase)

(ii) Elongation :

The elongation phase of replication includes two distinct but related events,
leading strand synthesis and lagging strand synthesis
Leading strand synthesis is more simple and begins with the synthesis of short (10 – 60 nucleotides) RNA primer by enzyme primase (Dna G protein)
at the DNA- Replication Deoxyribonucleotides are added to this primer by DNA pol III.

i. Leading strand synthesis:

Leading strand synthesis is a straightforward process which begins with the synthesis of RNA primer by primase at replication origin.
DNA polymerase III then adds the nucleotides at 3’end.
The leading strand synthesis then proceeds continuously keeping pace with unwinding of the replication fork until it encounters the termination sequences.

ii. Lagging strand synthesis:

The lagging strand synthesized in short fragments called Okazaki fragments.
At first RNA primer is synthesized by primase and as in leading strand DNA polymerase III binds to RNA primer and adds dNTPS.
On this level the synthesis of each okazaki fragments seems straight forward but the reality is quite complex.

Mechanism of Lagging strand synthesis

The complexity lies in the coordination of leading and lagging strand synthesis.
Both the strands are synthesized by a single DNA polymerase III dimer which accomplishes the looping of template DNA of lagging strand synthesizing Okazaki fragments.
Helicase (dnaB) and primase (dnaG) constitute a functional unit within a replication complex called primosome.
DNA pol III uses one set of core sub units (Core polymerase) to synthesize leading strand and another set of core subunit to synthesize lagging strand.
In elongation steps, helicase in front of primase and pol III, unwind the DNA at the replication fork and travel along lagging strand templates along 5’-3’ direction.
DnaG primase occasionally associated with dnaB helicase synthesizes short RNA primer. A new B-sliding clamp is then positioned at the primer by the B-clamp loading complex of DNA pol III.
When the Okazaki fragments synthesis is completed, the replication halted and the core sub unit dissociates from their sliding clamps and associates with new clamp.
This initiates the synthesis of new Okazaki fragments.
Both leading and lagging strand are synthesized co-ordinately and simultaneously by a complex protein moving in 5’-3’ direction.
In this way both leading and lagging strand can be replicated at same time by a complex protein that move in same direction.
Every so often the lagging strands must dissociates from the replicosome and reposition itself so that replication can continue.
Lagging strand synthesis is not completes until the RNA primer has been removed and the gap between adjacent Okazaki fragments are sealed.
The RNA primer are removed by exonuclease activity (5’-3’) of DNA pol-I and replaced by DNA. The gap is then sealed by DNA ligase using NAD as cofactor.

(iii) Termination :

Eventually the two replication forks of circular E. coli chromosomes meet at termination recognizing sequences (ter).
The Ter sequence of 23 bp is arranged on the chromosome to create a trap that the replication fork can enter but cannot leave. Ter sequences function as binding sites for TUS protein.
The Ter-TUS complex can arrest the replication fork from only one direction. Ter-TUS complex encounters first with either of the replication fork and halt it.
The other opposing replication fork halted when it collided with the first one.
This seems the Ter-TUS sequence is not essential for termination but it may prevent over replication by one fork if another is delayed or halted by a damage or some obstacle.
When either fork encountered the Ter-TUS complex, replication halted.
Final few hundred bases of DNA between these large protein complexes are replicated by not yet known mechanism forming two interlinked (catenated) chromosomes.
In E. coli DNA topoisomerase IV (type II) cut the two strands of one circular DNA and segrate each of the circular DNA and finally join the strand.
The DNA finally transferred to two daughter cells.

MCQs on DNA Replication

What is the origin of replication in E. coli called?
a) Ori R
b) Ori B
c) Ori C
d) Ori E
Answer: c) Ori C
How many base pairs long is the oriC sequence in E. coli?
a) 100 bp
b) 245 bp
c) 500 bp
d) 750 bp
Answer: b) 245 bp
How many 13 bp AT-rich repeat sequences are present in oriC?
a) 2
b) 3
c) 4
d) 5
Answer: b) 3
Which enzyme initiates the unwinding of DNA at the replication origin?
a) DNA ligase
b) DNA polymerase
c) DNA helicase
d) DnaA protein
Answer: d) DnaA protein
What is the role of DnaB helicase in DNA replication?
a) It seals the gaps between Okazaki fragments
b) It unwinds the DNA strands
c) It synthesizes RNA primers
d) It removes RNA primers
Answer: b) It unwinds the DNA strands
Which protein helps in the loading of DnaB onto DNA?
a) DnaC
b) DnaG
c) DNA gyrase
d) Tus protein
Answer: a) DnaC
What prevents the re-annealing of single-stranded DNA during replication?
a) DnaA protein
b) DNA ligase
c) Single-stranded binding protein (SSB)
d) DNA polymerase III
Answer: c) Single-stranded binding protein (SSB)
What is the function of DNA primase?
a) Synthesizes a short RNA primer
b) Unwinds DNA
c) Adds nucleotides to the growing strand
d) Seals the Okazaki fragments
Answer: a) Synthesizes a short RNA primer
Which enzyme is responsible for elongation in DNA replication?
a) DNA polymerase I
b) DNA polymerase III
c) DNA ligase
d) DNA helicase
Answer: b) DNA polymerase III
What are Okazaki fragments?
a) Continuous strands of DNA
b) Short DNA fragments synthesized on the lagging strand
c) DNA segments formed during transcription
d) RNA segments on the leading strand
Answer: b) Short DNA fragments synthesized on the lagging strand
Which enzyme removes RNA primers?
a) DNA helicase
b) DNA polymerase I
c) DNA polymerase III
d) DNA primase
Answer: b) DNA polymerase I
What is the direction of DNA synthesis?
a) 3’ → 5’
b) 5’ → 3’
c) Both directions simultaneously
d) Random
Answer: b) 5’ → 3’
Which enzyme seals the nicks between Okazaki fragments?
a) DNA polymerase I
b) DNA helicase
c) DNA ligase
d) Topoisomerase
Answer: c) DNA ligase
Which enzyme relaxes supercoiling ahead of the replication fork?
a) DNA polymerase III
b) DNA gyrase
c) DNA primase
d) DnaB helicase
Answer: b) DNA gyrase
What type of enzyme is DNA gyrase?
a) Helicase
b) Ligase
c) Topoisomerase
d) Polymerase
Answer: c) Topoisomerase
Which protein complex loads the β-sliding clamp?
a) DNA ligase
b) Clamp loader complex
c) DNA helicase
d) DNA primase
Answer: b) Clamp loader complex
What is the function of β-sliding clamp?
a) Increases DNA polymerase processivity
b) Binds RNA primers
c) Unwinds the DNA strands
d) Prevents DNA supercoiling
Answer: a) Increases DNA polymerase processivity
What is the function of Tus protein in replication termination?
a) Helps in primer synthesis
b) Prevents over-replication
c) Seals Okazaki fragments
d) Unwinds the DNA strands
Answer: b) Prevents over-replication
What is the length of the ter sequence in E. coli?
a) 10 bp
b) 15 bp
c) 23 bp
d) 30 bp
Answer: c) 23 bp
Which enzyme resolves the interlinked circular chromosomes after replication?
a) DNA polymerase III
b) DNA helicase
c) DNA topoisomerase IV
d) DNA ligase
Answer: c) DNA topoisomerase IV
How many core subunits does DNA polymerase III have?
a) 1
b) 2
c) 3
d) 4
Answer: b) 2
Which of the following is the primary function of DNA polymerase I?
a) Primer synthesis
b) Removal of RNA primers
c) Unwinding of DNA
d) Elongation of DNA strand
Answer: b) Removal of RNA primers
Which process describes the movement of the replication fork?
a) Unidirectional
b) Bidirectional
c) Multidirectional
d) Non-directional
Answer: b) Bidirectional
What is the role of primosome?
a) Unwinds DNA strands
b) Synthesizes RNA primers
c) Adds nucleotides to growing DNA strand
d) Prevents DNA supercoiling
Answer: b) Synthesizes RNA primers
Which enzyme is responsible for proofreading during replication?
a) DNA polymerase I
b) DNA polymerase III
c) DNA helicase
d) DNA primase
Answer: b) DNA polymerase III
In which direction does DNA helicase move along the lagging strand?
a) 3’ → 5’
b) 5’ → 3’
c) Randomly
d) Both directions
Answer: b) 5’ → 3’
What happens when the replication forks meet?
a) Replication speeds up
b) Replication terminates
c) Replication pauses temporarily
d) Replication reverses
Answer: b) Replication terminates
What is the function of the exonuclease activity of DNA polymerase I?
a) Seals Okazaki fragments
b) Removes RNA primers
c) Prevents supercoiling
d) Adds nucleotides
Answer: b) Removes RNA primers
The process of DNA replication is
a) Conservative
b) Semi-conservative
c) Dispersive
d) Random
Answer: b) Semi-conservative
What is the main purpose of the leading strand synthesis?
a) To create Okazaki fragments
b) To continuously synthesize DNA
c) To bind RNA primers
d) To unwind the DNA
Answer: b) To continuously synthesize DNA

Which enzyme prevents excessive supercoiling of DNA ahead of the replication fork?
a) DNA helicase
b) DNA topoisomerase
c) DNA primase
d) DNA polymerase III
Answer: b) DNA topoisomerase
DNA replication in prokaryotes occurs in which part of the cell?
a) Nucleus
b) Mitochondria
c) Cytoplasm
d) Endoplasmic reticulum
Answer: c) Cytoplasm
What is the function of DNA polymerase III’s 3′ → 5′ exonuclease activity?
a) Removes RNA primers
b) Proofreads newly synthesized DNA
c) Seals nicks in Okazaki fragments
d) Unwinds the DNA helix
Answer: b) Proofreads newly synthesized DNA
Which enzyme is responsible for the removal of supercoils introduced during replication?
a) DNA helicase
b) DNA ligase
c) DNA gyrase
d) DNA primase
Answer: c) DNA gyrase
How many replication forks are formed at the origin of replication?
a) 1
b) 2
c) 3
d) 4
Answer: b) 2
Which subunit of DNA polymerase III is responsible for proofreading?
a) Alpha
b) Beta
c) Epsilon
d) Omega
Answer: c) Epsilon
The lagging strand is synthesized in which direction?
a) 5′ → 3′
b) 3′ → 5′
c) Both directions
d) Randomly
Answer: a) 5′ → 3′
What is the function of the clamp-loading complex in DNA replication?
a) Synthesizes Okazaki fragments
b) Loads the β-sliding clamp onto DNA
c) Unwinds the DNA helix
d) Seals the gaps between fragments
Answer: b) Loads the β-sliding clamp onto DNA
In E. coli, which protein prevents the dissociation of DNA polymerase III from the template?
a) DnaA
b) β-sliding clamp
c) SSB protein
d) DNA ligase
Answer: b) β-sliding clamp
What happens to the RNA primers after DNA replication?
a) They remain in the DNA strand
b) They are converted into DNA
c) They are removed and replaced by DNA
d) They help in DNA ligation
Answer: c) They are removed and replaced by DNA
DNA replication in prokaryotes is
a) Bidirectional
b) Unidirectional
c) Non-directional
d) Multi-directional
Answer: a) Bidirectional
Which of the following is NOT involved in DNA replication?
a) DNA primase
b) RNA polymerase
c) DNA helicase
d) DNA polymerase III
Answer: b) RNA polymerase
What is the role of Tus protein?
a) Initiates replication
b) Loads the sliding clamp
c) Terminates replication
d) Unwinds DNA
Answer: c) Terminates replication
Which enzyme catalyzes the formation of the phosphodiester bond to seal nicks in DNA?
a) DNA helicase
b) DNA polymerase I
c) DNA ligase
d) Topoisomerase
Answer: c) DNA ligase
DNA polymerase III requires a primer because
a) It cannot initiate DNA synthesis on its own
b) It does not recognize single-stranded DNA
c) It functions only on RNA templates
d) It can synthesize only RNA, not DNA
Answer: a) It cannot initiate DNA synthesis on its own
What type of replication occurs in bacterial chromosomes?
a) Rolling-circle replication
b) Linear replication
c) Theta replication
d) Dispersive replication
Answer: c) Theta replication
What provides the energy for DNA polymerization?
a) ATP
b) GTP
c) dNTP hydrolysis
d) NAD+
Answer: c) dNTP hydrolysis
What happens when DNA polymerase encounters a damaged template?
a) It skips the damaged region
b) It stops replication permanently
c) It uses a bypass mechanism to continue replication
d) It repairs the DNA itself
Answer: c) It uses a bypass mechanism to continue replication
DNA replication is regulated to ensure
a) It occurs multiple times per cycle
b) It occurs only once per cell cycle
c) It happens randomly in the cell
d) It is delayed until cell division
Answer: b) It occurs only once per cell cycle
DNA polymerase III synthesizes DNA in which direction?
a) 3’ → 5’
b) 5’ → 3’
c) Both directions
d) Randomly
Answer: b) 5’ → 3’