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ProkaryoticDNAReplication–Enzymes,Steps
Published on: August 6, 2024 by
WhatisProkaryoticDNAReplication?
Prokaryotic DNA replication is a fundamental biological process where prokaryotes, such as
bacteria, duplicate their DNA to ensure genetic continuity during cell division. This intricate process
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Contents
What is Prokaryotic DNA Replication?
Structure of Ori C
Mechanism of prokaryotic dna replication
Enzymes involved in prokaryotic dna replication
Steps of DNA Replication
A. Initiation of Prokaryotic DNA Replication
B. Elongation of Prokaryotic DNA Replication
C. Termination of Prokaryotic DNA Replication
Other Prokaryotic replication models
Describe The Three-step mechanism of DNA ligation
Significance
FAQ
References
English
06/08/2024, 22:59 Prokaryotic DNA Replication - Enzymes, Steps - Biology Notes Online
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ensures that each daughter cell receives an accurate copy of the genetic material, crucial for their
survival and function.
DNA replication in prokaryotes follows a semi-conservative method. This means each newly
formed DNA molecule consists of one original parental strand and one newly synthesized daughter
strand. This method helps preserve the genetic information across generations.
Replication in prokaryotes, particularly well-studied in Escherichia coli (E. coli), begins at a unique
origin of replication known as OriC. Here, replication initiates bi-directionally, meaning it proceeds
in two opposite directions from the origin, facilitating efficient copying of the entire DNA molecule.
The process of prokaryotic DNA replication can be broken down into three main steps: initiation,
elongation, and termination.
During initiation, several proteins, including initiator proteins, bind to the OriC region, unwinding the
DNA to create replication forks. These forks are critical points where the DNA strands are
separated, allowing replication to begin.
Next, in the elongation phase, DNA polymerase enzymes add nucleotides to the growing DNA
strand, complementary to the template strand. This phase involves a complex interaction of
various proteins and enzymes, ensuring the accurate and rapid synthesis of the new DNA strands.
Finally, termination occurs when the replication forks meet, completing the replication process.
Specific termination sequences signal the end of replication, ensuring that the entire genome has
been accurately copied.
Prokaryotic DNA replication is an efficient and highly regulated process, critical for the survival and
proliferation of prokaryotic organisms. Understanding this process in detail provides valuable
insights into the fundamental mechanisms of life and can inform various applications in
biotechnology and medicine.
StructureofOriC
OriC (Origin of Chromosome) is the specific locus in Escherichia coli where DNA replication is
initiated. This critical region is located at approximately the 84.5 mpu position on the E. coli genome,
situated opposite to the replication termination site. The structure of OriC is characterized by two key
regions: the 9-mer and the 13-mer.
···

1. 9-mer Region (DNA-A Box):
Structure: The 9-mer is a conserved sequence of nine nucleotides found within OriC. This
region is also referred to as the DNA-A box.
Function: The 9-mer serves as the primary binding site for the DnaA protein, a key initiator of
DNA replication. DnaA binds specifically to this sequence, facilitating the formation of the DnaA-
oriC complex.
Role in Replication Initiation: Upon binding to the 9-mer, DnaA promotes the formation of a
multi-protein complex that is essential for unwinding the DNA helix and recruiting other
replication factors.
2. 13-mer Region:
Structure: The 13-mer consists of a repeating sequence of thirteen nucleotides characterized
by a high proportion of adenine-thymine (A-T) base pairs.
Function: This region is known for its lower melting temperature compared to regions with
higher guanine-cytosine (G-C) content.
Role in Replication Initiation: The high A-T content of the 13-mer facilitates the separation of
the DNA strands. This is crucial for the formation of the replication bubble, allowing access for
the replication machinery to initiate DNA synthesis.
Mechanismofprokaryoticdnareplication
The synthesis of a DNA molecule can be divided into three stages:
1. Initiation
2. Elongation
3. Termination
distinguished both by the reactions taking place and by the enzymes required. As you will find here
and in the next two chapters, synthesis of the major information containing biological polymers-DNAs,
RNAs, and proteins-can be understood in terms of these same three stages, with the stages of each
pathway having unique characteristics. The events described below reflect information derived
primarily from in vitro experiments using purified E col’i proteins, although the principles are highly
conserved in all replication systems.
···

Enzymesinvolvedinprokaryoticdnareplication
The enzymes involved in DNA replication are vital for ensuring the accurate duplication of the genetic
material. These enzymes each play a specific role, coordinating to achieve efficient and precise DNA
synthesis. Below is a detailed examination of the key enzymes and their functions during DNA
replication.
1. Helicases
Function: Unwind the DNA helix at the start of replication.
Mechanism: These enzymes break the hydrogen bonds between the DNA strands, creating
two single strands that serve as templates for replication.
2. Single-Strand Binding (SSB) Proteins
Function: Bind to the single strands of unwound DNA.
Role: Prevent reformation of the DNA helix during replication, stabilizing the single-stranded
DNA and protecting it from degradation.
3. Primase

Function: Synthesizes RNA primers.
Initiation: These RNA primers are necessary for the initiation of DNA chain synthesis, providing
a starting point for DNA polymerases to begin DNA synthesis.
4. DNA Polymerase III (DNAP III)
Function: Elongates the DNA strand.
Specifics: Adds deoxyribonucleotides to the 3′ end of the chain.
Directionality: Synthesis occurs in the 5′ to 3′ direction, which is a fundamental characteristic
of DNAP III’s function.
5. DNA Polymerase I (DNAP I)
Function: Replaces RNA primers with DNA.
Process: Removes RNA primers and fills in the gaps with the appropriate deoxynucleotides,
ensuring the continuity of the DNA strand.
6. DNA Topoisomerase I
Function: Relaxes the DNA helix during replication.
Action: Creates a temporary nick in one of the DNA strands to alleviate the tension generated
by the unwinding of the helix.
7. DNA Topoisomerase II
Function: Relieves the strain on the DNA helix.
Mechanism: Introduces supercoils into the DNA helix by creating nicks in both strands, thereby
preventing overwinding and tangling.
8. DNA Ligase
Function: Forms a 3′-5′ phosphodiester bond.
Role: Connects adjacent fragments of DNA, such as Okazaki fragments on the lagging strand,
ensuring the integrity and continuity of the newly synthesized DNA.
Enzyme Function Details
Helicases Unwind the DNA helix
Break hydrogen bonds
between DNA strands, creating
single strands for replication.
SSB Proteins
Bind to single strands of
unwound DNA
Stabilize single-stranded DNA
and prevent reformation of the
DNA helix during replication.
Primase Synthesizes RNA primers
Provides starting points for
DNA polymerases to initiate
DNA synthesis.
DNA Polymerase III Elongates DNA strand
Adds deoxyribonucleotides to
the 3′ end of the chain;
synthesizes in the 5′ to 3′
direction.
DNA Polymerase I
Replaces RNA primers with
DNA
Removes RNA primers and fills
in gaps with deoxynucleotides.

Enzyme Function Details
DNA Topoisomerase I Relaxes the DNA helix
Creates temporary nicks in one
DNA strand to alleviate tension
from unwinding.
DNA Topoisomerase II
Relieves strain on the DNA
helix
Introduces supercoils by
creating nicks in both DNA
strands to prevent overwinding
and tangling.
DNA Ligase
Forms 3′-5′ phosphodiester
bonds between DNA fragments
Connects adjacent DNA
fragments, ensuring the
continuity and integrity of the
newly synthesized DNA.
StepsofDNAReplication
Here is the detailed explanation of the steps involved in DNA replication:
···

1. Initiation at the Origin of Replication
Origin of Replication: DNA replication begins at a specific sequence known as the origin of
replication.
Unwinding of Double Helix: Enzymes such as helicase unwind the DNA double helix, creating
accessible single strands for replication.
2. Formation of Replication Forks
Replication Forks: Helicase activity generates a pair of replication forks at the origin, where
the DNA is actively unwound.
Stabilization: Single-Strand Binding (SSB) proteins stabilize the unwound DNA, preventing it
from re-forming the double helix. DNA topoisomerases alleviate the tension in the DNA helix by
creating temporary nicks.
3. Synthesis of RNA Primers
Primase Activity: Primase synthesizes short RNA primers (approximately 10 bases long) at
the replication forks.
Primer Role: These RNA primers provide the starting points for DNA polymerase to begin DNA
synthesis.
4. Elongation of the Leading Strand
Continuous Synthesis: DNA Polymerase III (DNAP III) elongates the leading strand
continuously in the 5′ to 3′ direction, following the replication fork.
5. Elongation of the Lagging Strand
Discontinuous Synthesis: The lagging strand is synthesized discontinuously, also in the 5′ to
3′ direction, through the formation of short DNA segments known as Okazaki fragments.
Fragment Synthesis: Multiple RNA primers are laid down by primase, allowing DNAP III to
synthesize Okazaki fragments.
6. Primer Removal and Replacement
DNA Polymerase I Activity: DNAP I removes RNA primers from both the leading and lagging
strands.
Gap Filling: DNAP I fills in the resulting gaps with the appropriate deoxynucleotides, ensuring
continuity of the DNA strand.
7. Sealing of DNA Fragments
DNA Ligase Function: DNA ligase seals the nicks between Okazaki fragments and the gaps
left after primer removal.
Formation of Continuous Strands: This sealing forms continuous and complete DNA strands,
finalizing the replication process.
A.InitiationofProkaryoticDNAReplication

Protein required for initiation of Replication in Prokaryotes
Protein required for initiation of Replication in Prokaryotes
Step1:Oric
The origin of replication in E. coli, known as oriC, is a critical region of the bacterial chromosome
where DNA replication begins. It spans 245 base pairs and contains specific DNA sequences essential

for initiating replication. The structure and function of oriC are well conserved among various bacterial
species, reflecting its fundamental role in the replication process. Below is a detailed examination of
the components and functions of oriC.
Prokaryotic DNA Replication: oric structure
Structure of oriC
Length and Composition: oriC is 245 base pairs long, comprising several specific sequence
motifs necessary for the initiation of DNA replication.
Key Sequence Elements
R Sites:
Definition: Five repeats of a 9-base pair sequence.
Function: Serve as binding sites for DnaA, the key initiator protein. DnaA binding is crucial
for the unwinding of the DNA at the origin.
DNA Unwinding Element (DUE):
Composition: A region rich in adenine-thymine (A) base pairs.
Function: The Aregion facilitates the unwinding of the DNA helix, as Abase pairs are less
stable than guanine-cytosine (G) pairs.
Binding Sites for Additional Proteins
I Sites:
Function: Specific binding sites for DnaA proteins, enhancing the binding affinity and
stability of the initiation complex.

IHF (Integration Host Factor):
Role: A DNA-binding protein that bends the DNA, facilitating the assembly of the initiation
complex.
FIS (Factor for Inversion Stimulation):
Role: Another DNA-binding protein that aids in the proper formation of the initiation
complex.
HU Protein:
Function: A histone-like bacterial protein that stabilizes the open complex formed during the
initiation of replication. Although it does not have a specific binding site, HU plays a
significant role in maintaining the structure necessary for replication.
Functional Overview
DnaA Binding: DnaA proteins bind to the R and I sites, causing the DNA to bend and partially
unwind at the DUE.
Assembly of Initiation Complex: IHF and FIS proteins further facilitate the bending and
stabilization of the DNA, promoting the formation of the open complex necessary for replication.
DNA Unwinding: The ADUE region is particularly susceptible to unwinding, allowing helicase
and other replication machinery to access and replicate the DNA strands.
Step2:OricRecognizebyDnaA
The initiation of DNA replication in E. coli is a complex process involving multiple proteins and
enzymes. Central to this process is the DnaA protein, a member of the AAA+ ATPase family, which
plays a critical role in recognizing the origin of replication, oriC. Below is a detailed explanation of the
steps involved in this recognition process.
···

1. Formation of the Pre-replication Complex
Protein Assembly:
At the onset of replication, at least 10 different enzymes and proteins assemble to open the
DNA helix.
This assembly sets up a complex required for subsequent replication steps.
2. Role of DnaA Protein
AAA+ ATPase Family:
DnaA is part of the AAA+ ATPase family, known for its role in various cellular activities.
It forms oligomers and hydrolyzes ATP at a slow rate, acting as a molecular switch.
Active vs. Inactive Forms:
The ATP-bound form of DnaA is active, while the ADP-bound form is inactive.
This ATP hydrolysis enables the protein to transition between active and inactive states.
3. Binding to oriC
Formation of DnaA Helix:
Eight ATP-bound DnaA molecules oligomerize to form a right-handed helical structure.
This structure wraps around the R and I sites in oriC.
Differential Binding:
DnaA has a higher affinity for R sites than I sites.
It binds to R sites equally well in both ATP and ADP-bound forms.
I sites bind exclusively to the ATP-bound form, distinguishing active DnaA.
4. Induction of Supercoiling
DNA Wrapping:
The DNA wraps tightly around the DnaA-oriC complex, creating a positive supercoil.
Effect on DUE Region:
The strain from the supercoiling destabilizes the adjacent ADNA unwinding element (DUE)
region.
This destabilization is critical for initiating DNA unwinding.
5. Involvement of Additional Proteins
···

DNA-Binding Proteins:
The complex at oriC also includes Hu, IHF (Integration Host Factor), and FIS (Factor for
Inversion Stimulation).
These proteins facilitate DNA bending, further promoting the formation of the replication
initiation complex.
Therefore, the recognition of oriC by DnaA involves a precise and coordinated sequence of events.
The assembly of the pre-replication complex, the ATP-dependent activation of DnaA, and the
differential binding to R and I sites are crucial steps. The induced supercoiling and the involvement of
additional DNA-binding proteins ensure that the DNA unwinding element is properly destabilized,
setting the stage for the initiation of DNA replication. This intricate process underscores the complexity
and efficiency of bacterial DNA replication mechanisms.
···

Initiation of Prokaryotic DNA Replication
Step3:DnaCProtein
In the intricate process of DNA replication, the DnaC protein plays a crucial role, particularly in the
loading of the DnaB helicase onto the DNA strands. This step ensures that the replication machinery
is properly assembled and ready for the synthesis of new DNA strands. Below is a detailed and
sequential explanation of how DnaC facilitates this process.
1. Binding of DnaC to DnaB
AAA+ ATPase Function:
Similar to DnaA, DnaC is a member of the AAA+ ATPase family.
Its primary role is to bind to the DnaB helicase and facilitate its attachment to the DNA
strands.
2. Formation of the DnaB-DnaC Complex
Ring-Shaped DnaB Helicase:
The DnaB protein forms a ring-shaped helicase structure.
This helicase forms a tight complex with six DnaC subunits, each bound to ATP.
Opening of the DnaB Ring:
The interaction between DnaC and DnaB opens the DnaB ring, allowing it to encircle the
DNA strands.
This process is further aided by an interaction between DnaB and the DnaA proteins already
bound at the origin of replication.
3. Loading of DnaB onto DNA Strands
Placement on DNA Strands:
One of the ring-shaped DnaB hexamers is loaded onto each of the single DNA strands
within the denatured DNA unwinding element (DUE) region.
This ensures that each DNA strand is primed for replication.
4. Hydrolysis of ATP
ATP Hydrolysis and Release of DnaC:
The ATP bound to the DnaC subunits is hydrolyzed, a reaction catalyzed by the presence of
water.
This hydrolysis releases the DnaC protein from the complex, but the DnaB helicase remains
tightly bound to the DNA.

SummaryofFunctionsandProcesses
1. DnaC Binding to DnaB:
Facilitates the interaction between DnaB and the DNA strands.
2. DnaB-DnaC Complex Formation:
Ensures the correct assembly of the helicase structure.
3. Loading DnaB onto DNA:
Positions the helicase for effective unwinding of the DNA helix.
4. ATP Hydrolysis:
Releases DnaC, leaving DnaB bound and functional on the DNA strands.
Therefore, DnaC acts as a crucial mediator in the assembly of the replication machinery, specifically
by loading the DnaB helicase onto the DNA. This step is essential for the continuation of the DNA
replication process, ensuring that the DNA strands are properly unwound and accessible for the
synthesis of new strands. The precise interaction and regulation by ATP hydrolysis underline the
complex yet highly coordinated nature of DNA replication.
Step4:RoleofDnaBProtein,SSB,andDNAGyraseinDNAReplication
The loading of the DnaB helicase is a pivotal step in the initiation of DNA replication. This step sets the
stage for the unwinding of the DNA helix and the establishment of replication forks. Here is a detailed
and sequential explanation of how DnaB, single-stranded DNA-binding proteins (SSB), and DNA
gyrase function in this process.
1. DnaB Protein: The Replicative Helicase
Unwinding DNA:
DnaB helicase is essential for unwinding the DNA double helix.
It moves along the single-stranded DNA in a 5′-to-3′ direction.
···

The two DnaB helicases bound to the DNA strands move in opposite directions, creating
two replication forks.
Interaction with Other Proteins:
DnaB is the central protein to which other replication fork proteins are connected.
It interacts with various components, including the τ subunits of the DNA polymerase III
holoenzyme, facilitating the polymerase’s function.
2. Single-Stranded DNA-Binding Protein (SSB)
Stabilizing Separated Strands:
As the DNA strands are separated at the replication fork, multiple SSB molecules bind to the
single-stranded DNA.
This binding stabilizes the separated strands, preventing them from reannealing or forming
secondary structures.
Protection and Prevention:
SSBs protect the single-stranded DNA from nucleases.
They also prevent the formation of hairpin loops, which can hinder the replication process.
3. DNA Gyrase (DNA Topoisomerase II)
Relieving Topological Stress:
DNA gyrase, a type of topoisomerase, alleviates the topological stress generated by the
unwinding of the DNA helix.
It introduces negative supercoils into the DNA, counteracting the positive supercoils formed
ahead of the replication fork.
Maintaining DNA Integrity:
By relieving the torsional strain, DNA gyrase ensures that the DNA remains intact and
accessible for replication machinery.
SummaryofFunctionsandProcesses
1. DnaB Protein:
···

Central Role: Unwinds the DNA double helix and facilitates the formation of replication forks.
Protein Interactions: Connects with other replication proteins to coordinate the replication
process.
2. Single-Stranded DNA-Binding Protein (SSB):
Stabilization: Binds to single-stranded DNA to stabilize and protect it.
Prevention: Prevents reannealing and secondary structure formation.
3. DNA Gyrase (DNA Topoisomerase II):
Stress Relief: Introduces negative supercoils to relieve topological stress.
Integrity Maintenance: Ensures DNA remains intact and accessible for replication.
RegulationofDNAReplicationInitiation
Control Mechanism:
The initiation of DNA replication is the only known regulated step, ensuring that replication
occurs only once per cell cycle.
Although the exact regulatory mechanisms are not fully understood, genetic and biochemical
studies have provided insights into several regulatory pathways.
Step5:ProteinHda
Once DNA polymerase III and the B subunits have been loaded onto the DNA, which marks the
end of the initiation phase, the protein Hda binds to the B subunits and interacts with DnaA to
speed up the hydrolysis of the ATP that is bound to DnaA.
Hda is also a AAA+ ATPase that is very similar to DnaA. (its name is derived from homologous to
DnaA).
This breakdown of ATP causes the DnaA complex at the beginning of the cell to fall apart.
The protein changes between its inactive (with bound ADP) and active (with bound ATP) forms
every 20 to 40 minutes. This happens because DnaA slowly lets go of ADP and rebinds ATP.
···

Step6: DNAadeninemethylation
Methylation of DNA and interactions with the bacterial plasma membrane can change when
replication starts.
The Dam methylase methylates the N6 spot of adenine in the palindromic sequence (5′)GATC,
which is part of the ori,C DNA. (DNA adenine methylation is what “Dam” means in biochemistry.)
The E. coli ori,C region has a lot of GATC sequences. It has 11 of them in 245 bp, while the
average number of GATC sequences in the whole E. coli chromosome is 1 in 256 bp.
The DNA is hemimethylated right after replication. The parent strands have methylated ori,C
sequences, but the new strands don’t. The protein SeqA now binds to the hemimethylated oriC
sequences and keeps them from moving. This is done by the interaction with the plasma
membrane (how this happens is not known).
After some time, ori,C is released from the plasma membrane, SeqA breaks apart, and the DNA
must be fully methylated by Dam methylase before it can bind to DnaA again and start a new
round of replication.
B.ElongationofProkaryoticDNAReplication
During the elongation phase of replication, Ieading strand synthesis and Lagging strand synthesis
take place. These are two separate but related processes.
At the replication fork, there are several enzymes that are important for making both strands. First,
DNA helicases unwind the parent DNA. Then, topoisomerases relieve the topological stress that is
caused.
Then, SSB is used to keep each strand in place. Synthesis of the leading and lagging strands is
very different from this point on.
Leadingstrandsynthesis
··· ···

The simpler of the two processes, leading strand synthesis starts with primase (DnaG protein)
making a short (10–60 nucleotide) RNA primer at the replication origin.
DnaG and DnaB helicase work together to make this reaction happen. The primer is made in the
opposite direction that the DnaB helicase is moving.
In reality, the DnaB helicase moves along the strand that becomes the lagging strand during DNA
synthesis. However, the first primer that is laid down during the first DnaGDnaB interaction starts
DNA synthesis on the leading strand in the opposite direction.
A DNA polymerase III complex linked to the DnaB helicase on the other DNA strand adds
deoxyribonucleotides to this primer.
Then, the process of making the leading strand keeps going at the same rate as the unwinding of
DNA at the replication fork.
··· ···

Synthesis of Okazaki fragment
Laggingstrandsynthesis
We’ve already said that lagging strand sythesis is done in short Okazaki fragments.
First, primase makes an RNA primer. Then, just like in leading strand synthesis, DNA polymerase
III binds to the RNA primer and adds deoxyribonucleotides.

On this level, putting together each Okazaki fragment seems easy, but it’s actually quite hard. The
coordination of leading and lagging strand synthesis is what makes it hard.
A single asymmetric DNA polymerase III dimer makes both strands. This is done by looping the
DNA of the Lagging strand, which brings the two points of polymerization together.
SynthesisofOkazakifragment
The synthesis of Okazaki fragments is a crucial aspect of DNA replication on the lagging strand. This
process involves a complex series of enzymatic activities, orchestrated with precision to ensure
accurate DNA duplication.
1. Formation of Primosomes:
Primosomes are key functional units within the replication complex. They consist of two
essential components: DnaB helicase and DnaG primase. DnaB helicase is responsible for
unwinding the DNA double helix at the replication fork, while DnaG primase synthesizes short
RNA primers necessary for DNA polymerase activity.
2. Role of DNA Polymerase III:
DNA Polymerase III plays a central role in DNA replication. Its core polymerase subunits are
continuously engaged in synthesizing the leading strand. On the lagging strand, however, the
core polymerase subunits cycle from one Okazaki fragment to the next. This movement is
facilitated by the formation of a looped structure in the lagging strand template.
3. Unwinding and Primer Synthesis:
As DNA Polymerase III progresses along the lagging strand in the 5′ to 3′ direction, DnaB
helicase unwinds the DNA in front of it. In certain instances, DnaG primase collaborates with
DnaB helicase to generate a short RNA primer. This primer serves as the starting point for DNA
synthesis.
4. Clamp Loading and Synthesis:
···

Following primer synthesis, the clamp loading complex of DNA Polymerase III deposits a new
β sliding clamp onto the primer. This clamp is essential for the stability and processivity of
DNA polymerase during the elongation phase.
5. Detachment and Re-association:
Upon completing an Okazaki fragment, the core subunits of DNA Polymerase III detach from
the β sliding clamp and the newly synthesized fragment. These subunits then re-associate with
a new β sliding clamp to initiate the synthesis of the next Okazaki fragment.
6. Function of the Replisome:
The replisome is a complex of proteins located at the replication fork, playing a critical role in
coordinating DNA synthesis. It integrates the activities of various enzymes, ensuring the
efficient and accurate replication of DNA.
mechanism of prokaryotic dna replication

···

TheclampLoadingcomplexofDNApolymeraseIII
The clamp Loading complex of DNA polymerase III, consisting of parts of the two τ subunits along
with the γ, δ, and δ’ subunits, is also an AAA+ ATPase.
Together, ATP and the novel B sliding clamp form a stable combination. The dimeric clamp
undergoes strain as a result of the binding, and the ring opens up at one of the subunit interfaces.
Through this rip, the newly primed lagging strand is introduced to the ring.
Hydrolysis of ATP by the clamp loader then allows the B sliding clamp to close around the DNA.
···

Beta sliding clamp
DNAsynthesis
The replisome is responsible for the rapid synthesis of DNA, at a rate of -1,000 nucleotides per
second per strand (leading and lagging).
DNA ligase seals the residual nick in an Okazaki fragment after DNA polymerase I has removed
the RNA primer.
DNA ligase is an enzyme that catalyses the creation of a phosphodiester link between a 3′
hydroxyl and a 5′ phosphate on opposite ends of DNA strands.
Adenylylation is required to activate the phosphate. The ATP is used by DNA ligases that have
been purified from viruses and eukaryotes.
Bacterial DNA ligases are distinct in that many of them derive the AMP activating group from the
coenzyme NAD+, which typically catalyses hydride transfer events. DNA ligase is another DNA
metabolic enzyme that has emerged as a crucial tool in recombinant DNA research.
···

Proteins required for replisome
Proteins required for replisome
C.TerminationofProkaryoticDNAReplication
final step of DNA Synthesis
Ultimately, the circular DNA replication split in two. The two halves of an E. coli chromosome join at
a terminus region called Ter, which contains multiple copies of a 20-base-pair sequence.

The Ter sequences on the chromosome are organised in a way that traps a replication fork inside.
Protein Tus binds to specific sequences in the Ter RNA (terminus utilisation substance).
Only one way of a replication fork can be stopped by the Tus-Ter complex. Each replication cycle
only allows one Ttrs-Ter complex to work, and that is the complex that is encountered first by one
of the two replication forks. Ter sequences may prevent over replication by one fork in the case
that the other is delayed or halted by encountering DNA damage or another impediment, even
though opposing replication forks typically halt when they intersect.
So, replication stops when one of the forks runs into a working Tus-Ter complex, and the other fork
stops when it runs into the first (arrested) fork.
Following replication of the last few hundred base pairs of DNA (by a mechanism we don’t fully
understand), two topologically connected (catenated) circular chromosomes are formed.
Catenanes are the name given to interconnected DNA cyclins.
The enzyme topoisomerase IV is necessary for the separation of the catenated rings in E. coLi (a
type II topoisomerase).
Once the chromosomes have been untangled, they are distributed evenly throughout the two
daughter cells.
Many DNA viruses that infect eukaryotic cells undergo a similar process near the end of their
replication cycle, which is also characteristic of other circular chromosomes.
···

Termination of chromosome Replication in E .coli
Roleoftopoisomerasesinreplicationtermination
During the final stages of DNA replication, the chromosomes are interlinked as catenanes, which are
topologically entangled circles of DNA. These catenanes form as a result of the separation of the
opposing replication forks. The resolution of these interlinked structures is crucial for ensuring the
accurate segregation of chromosomes into daughter cells. Topoisomerases, particularly DNA
topoisomerase IV, play a pivotal role in this process. Below is an in-depth explanation of their
functions.
1.FormationofCatenanes
Definition and Structure:
Catenanes are structures where two or more DNA molecules are interlinked in a manner similar
to interlocking rings.
These structures arise from the process of DNA replication when the newly synthesized strands
of DNA from each fork become intertwined.
Formation Mechanism:
During replication, the two replication forks move in opposite directions along the DNA
molecule.
As they proceed, they complete the synthesis of new DNA strands, resulting in the formation of
interlinked catenanes.
2.FunctionofTopoisomerasesinResolution
Topoisomerase IV:
Type II Topoisomerase:
DNA topoisomerase IV is a type II topoisomerase found in E. coli and other bacteria.
It facilitates the resolution of catenanes by transiently breaking both strands of one DNA
molecule, allowing the passage of the other linked molecule through the break.
Mechanism of Action:
Breaking and Rejoining:

Topoisomerase IV introduces transient double-strand breaks in one of the catenated
chromosomes.
This temporary cleavage allows the interlinked chromosome to pass through the break.
After the passage, the enzyme reseals the breaks, thus resolving the catenane structure.
Functional Importance:
Prevents Chromosome Entanglement:
By resolving catenanes, topoisomerase IV ensures that the chromosomes are separated
into individual, unlinked entities.
This is crucial for proper chromosome segregation during cell division.
Facilitates Accurate Cell Division:
The removal of interlinked chromosomes ensures that each daughter cell receives a
complete and accurate set of chromosomes.
Role in Eukaryotic Cells:
Topoisomerase II:
In eukaryotic cells, a similar role is performed by topoisomerase II.
This enzyme also resolves catenanes and other topological constraints that arise during
DNA replication and chromosome segregation.
3.ImplicationsforCellularProcesses
Chromosome Segregation:
Effective resolution of catenanes is essential for accurate chromosome segregation.
Errors in this process can lead to chromosome missegregation and genomic instability.
Replication Termination:
The resolution of catenanes marks the completion of the replication process.
This step is critical to ensure that replication termination is achieved and that the newly
synthesized chromosomes are properly prepared for cell division.
Drug Targets:
···

Antibiotic and Anticancer Therapies:
Topoisomerases, including topoisomerase IV, are targets for antibiotics and anticancer
drugs.
Inhibitors of these enzymes can effectively disrupt DNA replication and cell division in
bacteria and cancer cells.
Summary
Topoisomerase IV is crucial in resolving catenanes by transiently breaking and rejoining DNA
strands, thereby ensuring proper chromosome segregation and the completion of DNA replication.
Topoisomerases facilitate the disentangling of interlinked chromosomes, preventing replication
errors and maintaining genomic stability.
Understanding the role of topoisomerases provides insights into cellular processes and highlights
potential therapeutic targets for managing bacterial infections and cancer.
Role of topoisomerases in replication termination
OtherProkaryoticreplicationmodels

The duplication of theta type has already been mentioned. Rolling-circle replication and D-loop
replication are two further forms of bacterial reproduction.
RollingCircleReplication
The same circular template DNA spins during bacterial conjugation, and it is around this DNA that
a new strand of DNA forms.
The relaxase enzyme forms a nick in one strand of the conjugative plasmid at the oriT when
signalling initiates conjugation.
Relaxase can function either independently or as part of a larger complex of over a dozen proteins
called a relaxosome.
Relaxase enzyme TraI, along with its cofactors TraY and TraM, and the integrated host factor IHF,
make up the relaxosome of the F-plasmid system.
Following its nicking, the T-strand is unravelled from the intact strand and transmitted to the
recipient cell in a 5′-terminus-to-3′-terminus manner.
In either case, the residual strand undergoes replication, with or without the help of conjugation
(conjugative replication similar to the rolling circle replication of lambda phage).
It’s possible that a second nick is necessary for effective transfer during conjugative replication.
The second nicking event is supposedly blocked by compounds that imitate an intermediate step
of the process, according to a recent paper.

Madprime, CC BY-SA 4.0, via Wikimedia Commons
D-loopreplication
Organellar DNA is the most common type of DNA to undergo D-loop replication, which results in
the formation of a displacement loop, a triple stranded structure.

DescribeTheThree-stepmechanismofDNAligation
1. DNA ligase I is a cellular enzyme that catalyses the covalent ligation of adenosine monophosphate
(AMP) to DNA via hydrolysis of adenosine triphosphate (ATP).
2. Following a nick in duplex DNA, the ligase polypeptide transfers an AMP group to the nick’s 5′
phosphate termini.
3. In a process involving nucleophilic assault by the 3’HO group and release of AMP, the non-
adenylated ligase catalyzes the creation of the phosphodiester link.
···

The Three-step mechanism of DNA ligation | Image Source: Howes, Timothy & Tomkinson, Alan. (2012). DNA ligase I, the r
eplicative DNA ligase. Sub-cellular biochemistry.
Significance
DNA replication is a critical genetic process essential for cell growth, division, and the conservation of
genetic information. This process ensures that each new cell receives an accurate copy of the
genome, facilitating proper cellular function and inheritance. Here, we explore the significance of DNA
replication in a detailed and sequential manner.
···

Foundation of Genetic Continuity
Genome Conservation:
DNA replication preserves the entire genome, ensuring genetic information is accurately
passed to the next generation.
This conservation is vital for maintaining the integrity of the species’ genetic blueprint.
Inheritance:
Each daughter cell receives an identical copy of the DNA, preserving genetic traits and
characteristics through generations.
Cell Growth and Division
Regulation of Cell Cycle:
DNA replication is tightly regulated to coincide with the cell cycle, ensuring cells only divide
when they have a complete set of genetic instructions.
Proper timing of replication prevents mutations and genomic instability.
Mitotic and Meiotic Division:
During mitosis, DNA replication allows for the production of two genetically identical
daughter cells, essential for growth and tissue repair.
In meiosis, replication precedes genetic recombination and reduction division, crucial for
sexual reproduction and genetic diversity.
Molecular Mechanisms and Fidelity
Enzymatic Precision:
DNA polymerases and other replication enzymes ensure high-fidelity synthesis of new DNA
strands, minimizing errors.
···

Proofreading mechanisms correct any mismatches, enhancing the accuracy of replication.
Replication Fork Dynamics:
The replication fork, where DNA unwinding and synthesis occur, is orchestrated by a suite of
proteins, including helicases, primases, and ligases.
These proteins ensure the replication process proceeds smoothly and efficiently.
Impact on Cellular Function
Protein Synthesis:
Accurate DNA replication ensures the correct sequence of genes, which is crucial for the
synthesis of functional proteins.
Proper protein synthesis underlies all cellular functions, from metabolism to signaling.
Cellular Differentiation:
Replication fidelity allows cells to differentiate correctly, enabling the development of
specialized tissues and organs.
Biological Implications
Development and Growth:
From a single fertilized egg, DNA replication drives the development of a multicellular
organism by enabling successive cell divisions.
It supports the growth and maintenance of tissues throughout an organism’s life.
Repair and Regeneration:
DNA replication plays a role in repairing damaged DNA, maintaining genomic stability, and
allowing tissues to regenerate after injury.
FAQ
DNA replication in prokaryotes is called theta (theta) replication because this DNA is circular in
shape.
Prokaryotic DNA replication is the process by which prokaryotic organisms duplicate their DNA to
ensure accurate transmission of genetic information to daughter cells.
Whatisthereplicationofdnainprokaryoticcellscalled? ?
WhatisprokaryoticDNAreplication? ?
···

Prokaryotic DNA replication consists of three steps: initiation, elongation, and termination.
Initiation begins at a specific site called the origin of replication (OriC), where proteins bind to the
DNA sequence and helicase unwinds the DNA double helix.
During elongation, DNA polymerase III adds nucleotides one by one to the growing DNA chain,
using the parental DNA strands as templates.
DNA polymerase III is the primary enzyme responsible for synthesizing the new DNA strands by
adding nucleotides in the 5′-3′ direction.
Termination occurs when the replication forks meet at specific termination sites on the DNA
molecule, leading to the separation of the newly synthesized DNA strands.
OriC is a specific DNA sequence where replication begins. It serves as a recognition site for
proteins that initiate the replication process.
WhatarethethreemainstepsinvolvedinprokaryoticDNAreplication? ?
HowdoesinitiationofprokaryoticDNAreplicationoccur? ?
WhathappensduringelongationinprokaryoticDNAreplication? ?
WhatistheroleofDNApolymeraseIIIinprokaryoticDNAreplication? ?
HowisterminationofprokaryoticDNAreplicationachieved? ?
Whatisthesignificanceoftheoriginofreplication(OriC)inprokaryoticDNA
replication?
?

Helicase plays a crucial role in prokaryotic DNA replication by unwinding the DNA double helix,
separating the parental strands and creating replication forks.
RNA primers, synthesized by the enzyme primase, provide a starting point for DNA synthesis by
DNA polymerase. They serve as templates for the addition of DNA nucleotides.
DNA ligase seals the gaps between Okazaki fragments, which are short DNA segments
synthesized on the lagging strand, creating a continuous DNA strand.
References
Howes, Timothy & Tomkinson, Alan. (2012). DNA ligase I, the replicative DNA ligase. Sub-cellular
biochemistry. 62. 327-41. 10.1007/978-94-007-4572-8_17.
https://www.sciencedirect.com/science/article/pii/S0021925819820197
https://openoregon.pressbooks.pub/mhccmajorsbio/chapter/dna-replication-in-prokaryotes/
https://chem.libretexts.org/Courses/Brevard_College/CHE_301_Biochemistry/06%3A_Nucleic_Aci
ds/6.06%3A_DNA_Replication_in_Prokaryotes
https://pressbooks-dev.oer.hawaii.edu/biology/chapter/dna-replication-in-prokaryotes/
https://openstax.org/books/biology-2e/pages/14-4-dna-replication-in-prokaryotes
https://bitesizebio.com/10279/how-dna-ligation-works/
···
WhatisthefunctionofhelicaseinprokaryoticDNAreplication? ?
HowareRNAprimersinvolvedinprokaryoticDNAreplication? ?
WhatistheroleofDNAligaseinprokaryoticDNAreplication? ?

https://unacademy.com/content/neet-ug/study-material/biology/dna-replication-process-in-
prokaryotes/
https://wou.edu/chemistry/courses/online-chemistry-textbooks/ch450-and-ch451-biochemistry-
defining-life-at-the-molecular-level/chapter-9-dna-replication-and-repair-2/
https://geneticeducation.co.in/prokaryotic-dna-replication-initiation-elongation-and-termination/
https://www.texasgateway.org/resource/144-dna-replication-prokaryotes
https://thebiotechnotes.com/2019/07/13/dna-replication-in-prokaryotes/
https://en.wikipedia.org/wiki/Prokaryotic_DNA_replication
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