DNA REPLICATION IN PROKARYOTES

1. DNA Replication in
Prokaryotes
Submitted to:- Dr.Anupam Jyoti
Submitted by:- Dipak Kumar Singh
Puranjaya Pancholi

2. Introduction
DNA replication is the biological process of
producing two identical replicas of DNA from
one original DNA molecule.
It is a biological process occurring in all living
organisms and it is the basis for biological
inheritance.
It is a semi-conservative process which was
proved by Meselson and Stahl.

3. Some general features of DNA
replication
The very first step in DNA replication is to
‘unzip’ the double helix structure of
the DNA molecule.
This is carried out by an enzyme called
“Helicase” which breaks the hydrogen
bonds holding the complementary bases of
DNA together (A with T, C with G).

4. Contd….
Helicase separates the DNA to form a
replication fork at the origin of replication
where DNA replication begins.
One of the strands is oriented in the 3’ to 5’
direction (towards the replication fork), this is
the leading strand. The other strand is
oriented in the 5’ to 3’ direction (away from the
replication fork), this is the lagging strand.

5. Contd….
Replication forks extend bi-directionally as
replication continues.
Okazaki fragments are formed on the lagging
strand, while the leading strand is replicated
continuously.
“DNA ligase” seals the gaps between the Okazaki
fragments.
Primase synthesizes an RNA primer with a free 3′-
OH, which DNA polymerase III uses to synthesize
the daughter strands.

6. Process of DNA replication
DNA replication completes in various steps:
1. Initiation: Replication begins at an “origin of
replication”
2. Elongation: New strands of DNA are
synthesized by DNA polymerase.
3. Termination: Replication is terminated
differently in prokaryotes and eukaryotes.

7. 1. Initiation:
The chromosome of a prokaryote is circular
molecule of DNA.
Replication begins at one origin of replication
and proceeds in both directions around the
chromosomes.

8. Contd….
The enzymes involved in the DNA replication are
closely associated in one large complex called
replisome .
The replisome consists of :
• The primosome – composed of primase and
helicase.
• DNA polymerase III molecules.
• Topoisomerase, ligase, SSB, initiator proteins.

10. Activities take place in this
step:
The very beginning step of DNA replication is unwinding of
DNA molecules. This is done by the enzyme DNA helicase
which cause the two parent DNA strands to unwind and
separate from one another at the “origin of replication” to
form two Y- shaped replication forks.
“These replication forks are the actual site of DNA
copying”.

11. Contd….
After this, Helix destabilizing proteins binds to
the single stranded regions so that the two
strands do not rejoin.
Enzymes called topoisomerase produce breaks
in the DNA and then rejoins them in order to
relieve the stress in the helical molecule during
replication.

12. 2. Elongation :
Activities take place in this step:
In this phase, the synthesis of two new daughter
strand takes place complementary to the template
strand.
DNA polymerase III is the enzyme that
synthesizes the daughter strands.
At this point, a primer is needed so that DNA
polymerase III can begin to act.
This phase is marked by the synthesis of leading
strand and lagging strand.

14. Contd….
Leading strand is synthesized continuously in
5’ to 3’ direction along the direction of the
movement of replication fork.
Lagging strand synthesis occurs
discontinuously by loop formation in short
segments called Okazaki fragments.

15. Contd….
The lagging strand is looped so that DNA synthesis
proceeds steadily on both the leading and lagging
strand templates at the same time.
The synthesis of Okazaki fragments on the lagging
strand requires DnaB helicase and DnaG primase
that constitute a functional unit within the replication
complex, the primosome.
DnaB helicase first unwinds the replication forks.

16. Contd….
DNA primase then associates with DnaB
helicase, which synthesizes a short RNA
primer.
The clamp loading complex of DNA pol III
loads a beta- sliding clamp to the primer.
The primer is then extended by the DNA pol
III, which completes the synthesis of one
Okazaki fragment.

18. Contd….
Once an Okazaki fragment has been
completed, it’s RNA primer is removed and
replaced with DNA by DNA pol I, and the
remaining nick is sealed by DNA ligase.
DNA ligase catalyses the formation of
phosphodiester bond.

19. 3. TERMINATION
Activities take place in this step:
Replication of bacterial genome proceeds bi-
directionally which terminates at a position
diametrically opposite to the origin of
replication.
Replication terminates at the terminus region
containing multiple copies of a 20bp
sequence called Ter (terminus) sequences.

20. Contd….
Ter sequence works as a binding site for
protein Tus (terminus utilization substance)
which stops the DnaB helicase, resulting in
the termination of DNA replication.
The completed chromosomes then partitioned
into two daughter cells during cell division.

22. Important enzymes/proteins for
DNA replication

23. DNA POLYMERASE
DNA polymerase is an enzyme that carries out the
synthesis of a new strand on the template strand.
It is found in both prokaryotes and eukaryotes.
Three types of DNA polymerase takes part in
prokaryotic DNA replication, they are:
a) DNA polymerase I
b) DNA polymerase II
c) DNA polymerase III

24. DNA Polymerase I
It is the first DNA polymerase to be isolated
and purified.
DNA polymerase I has 5’- 3’ exonuclease
activity.
The 5’- 3’ exonuclease activity allows the
removal of ribonucleotides of the RNA primer,
utilized to initiate DNA synthesis.
The 5’- 3’ exonuclease activity is also utilized
during the repair of damaged DNA.

25. DNA Polymerase II
It has no 5’- 3’ exonuclease activity, rather it
has 3’- 5’ exonuclease activity.
The 3’- 5’ exonuclease function is utilized
during replication to allow DNA polymerase to
remove mismatched bases and is referred to
as the proof-reading activity of DNA
polymerase.
It is also utilized during the repair of damaged
DNA.

26. DNA Polymerase III
DNA polymerase III is the principle replication
enzyme required for DNA synthesis.
Prokaryotic DNA is replicated by DNA
polymerase III in the 5′ to 3′ direction at a rate
of 1000 nucleotides per second.
DNA polymerase III has very high processivity
and therefore, synthesizes DNA very quickly.

27. Conclusion
DNA replication is semi-conservative, with
each existing strand serving as a template for
the synthesis of a new strand.
Replication begins at, and proceeds bi-
directionally from the point of origin.
On one strand, synthesis is continous and on
the other strand the synthesis is discontinous.

28. Contd….
Okazaki fragments are joined together by
DNA ligase.
DNA polymerase III is the main replication
enzyme, DNA polymerase I is responsible
for special functions during replication and
repair.
Ter sequence works as the binding site for
protein Tus (terminus utilization substance)
which stops the DnaB helicase, resulting in
termination of DNA replication.