Easy point of DNA replication in prokaryotes

2. Presentation of Biochemistry
Assigned by : Mam Fatima
Outline
# DNA replication in prokaryotes
# DNA replication in eukaryotes

3. Participants
• SHUMAILA SHAREEF
• RANA M. FURQAN
• SHAHZEEN GHAFFAR
• KHURRUM MEHBOB
• ANEEZA YOUSAF
• LAILA RUBAB
• BISMA BIBI
• TEHMEENA ABID

4. DNA replication in prokaryotes
 DNA replication is a biological process which
produces two identical daughter DNA strands
from a double stranded parental DNA.
 A basis for biological inheritance in all living
organisms

5. Prokaryotic DNA replication
 DNA replication is semi conservative, each strand
is used as template for the synthesis of new
complementary strand.
 It is bidirectional, the y shaped formation of
replication fork running in opposite directions.
 It is semi discontinuous, the leading strand copies
continuously and the lagging strand copies in
segments(Okazaki fragments).

6. Replicon
⚫ Replicon is a DNA segment that undergoes replication.
⚫ It consists of an origin where replication begins and a
terminus where replication stops.
E. coli has a single replicon on its chromosome, as do
most prokaryotes.

7. Origin of replication
• Replication is initiated at the origin of replication
called Ori C which is 245 base pairs long and rich in
AT sequences.
• This sequence of bps is recognized by a protein
that binds to this site and opens up the DNA.
• An enzyme, helicase unwinds the DNA by breaking
the
hydrogen bonds between nitrogenous bps.
⚫ ATP hydrolysis is required for this process, the
energy released by it is used for the break down of
hydrogen bond.

8. Bidirectional replication
As the DNA opens up, two Y-
shaped structures called replication
forks are formed at the origin of
replication making a replication
bubble.
• The replication forks get extended
bidirectionally/opposite directions as
the replication proceeds.
⚫ Single stranded binding proteins
coat the single strand of DNA near
the replication fork to prevent single
stranded DNA from winding back
into double helix.

9. DNA polymerases
 DNA polymerases refers to a group of enzymes
that is responsible for DNA synthesis.
 The DNA polymerases have an additional activity
that is nuclease activity, the ability to break
phosphodiester bonds between nucleotides .
 All the DNA polymerases synthesize
polynucleotides in 5' to 3' direction and requires a
primer.

10. Prokaryotic DNA polymerases
 In prokaryotes, three main types of Polymerases are
known as DNA pol I, DNA pol II, and DNA pol III.
 DNA pol I has a exonuclease activity, which serves
as proofreading function to remove a mispaired base
and removes primarase after nucleotides addition.
 DNA pol II, doesn't play role in replication, is
involved in DNA repair Processes.
 DNA pol III is the main replication enzyme
responsible for the bulk DNA synthesis; it adds
nucleotides one by one to the growing DNA chain.

11. Replication fork –leading strand
• As we know DNA is antiparallel, that means one
strand runs in the 3' to 5' direction and its
complementary strand runs in 5' to3' direction.
• While the polymerases can only extend in 5' to 3'
direction which poses a slight problem at replication
fork.
⚫ One strand( 5' to 3') which is complementary to 3'
to 5' parental DNA strand is synthesized continuously
towards the replication fork because the polymerase
can add nucleotides in this direction.
⚫ This continuously strand is known as leading
strand.

12. Lagging strand
The other strand complementary to 5' to 3' parental
DNA is extended away from the replication fork in
small segments known as Okazaki Fragments, each
requiring a primer to start synthesis.
• The strand with Okazaki fragments(named after
the scientist who discovered it) is known as lagging
strand.

14. Enzymes and proteins involved in
replications
Helicase opens up the DNA at the replication fork.
⚫ Single-strand binding proteins coat the DNA
around the replication fork to prevent rewinding of
the DNA.
• Topoisomerase works at the region ahead of the
replication fork to prevent supercoiling.

15. Primase synthesizes RNA primers complementary to
the DNA strand.
⚫ DNA polymerase III extends the primers, adding
on to the 3' end, to make the bulk of the new DNA.
⚫ RNA primers are removed and replaced with DNA
by DNA polymerase I.
⚫ The gaps between DNA fragments are sealed by
DNA ligase.

17. Initiation
Initiation means to start or to initiate something.
During prokaryotic DNA replication the protein Dna A
(chromosomal replication initiator protein) bind to
the origin of replication while Dna B helicase unwinds
the DNA helix and two replication forks are formed at
the origin of replication
It is the bidirectional replication.

18. The main events involved in replication
initiation
• Recognition of origin.
• DNA melting.
⚫ Stabilization of single strand.
• Assembly of Primosome at the two forks produced.
Start synthesis of two daughter strands.

19. Proteins for initiation
Replication initiation in E.coli requires 6 proteins.
⚫ Dna A or chromosomal replication initiator protein
⚫ Dna B or replicative DNA helicase
Dna C (DNA replication protein)
SSBP (single stranded DNA-binding protein)
⚫ DNA gyrase
⚫ Dna G primase

20. Recognition of origin
Genetic studies ( in prokaryotic ) suggested that
initiation of replication at oriC most likely depend on
the protein encoded by a gene designated DnaA,
DnaB, DnaC .

21. DNA denaturation
2 to 4 DnaA protein binds to the 9 mer sequences called R
boxes to DAR(DnaA Assembly region).DNA coils around the
protein which induces the topological stress cause
Denaturation of AT rich that is DUE (DNA un winding)region
at 13 mer site to the left.
An aggregate having 6 molecules each of DnaB and DnaC are
formed.
⚫ The DnaC (helicase loader) loads the DnaB to the DUE site
then itself getdetach. The process is called helicase loading.
⚫ Then DnaB move and break hydrogen bond of AT rich
region.
⚫ DnaB functions as helicase and begins to unwind the DNA.
⚫ Gyrase facilitates unwinding by helicase as it provides a
swivel.

22. Stabilization of single strand
SSBP (single stranded DNA binding
protein)
SSBP healthy protein is necessary
which binds to single strand regions
for stabilization and also to reduce
them from reannealing.
Initiation of replication generally
requires ~ 60bp of unwound DNA
and the process consumes ATP.
⚫ One DnaB hexamer binds to each
of the two forks produced by
unwinding at the origin.

23. Assembly of primosome at the teo forks
Once a replication fork is generated,primosome assembles
at the origin,and initiates primer synthesis this is called
priming.
• Priming occurs only once and at the origin for the
replication of the leading strand.
⚫ But for replication of the lagging strand, priming occurs
repeatedly at intervals of 1000 to 2000 bases.
• Priming reaction at oric is rather simple the primosome
consists of a single protein, DnaG.
• DnaG needs to be activated by DnaB. DnaB also serves as
helicase, while DnaG carries out primer synthesis, primers
of15-50 bases are normally synthesized.
⚫ The replication fork proceeds in the 5 to 3 direction in
relation to the lagging strand

24. The replication fork advances and generates a single
stranded region of the lagging strand bound to SSBP
ahead of the primosomes. The primosomes moves
along this single stranded region.
. When the primosome reaches a site at which
priming can occur, it synthesis an RNA primer. This
primer sponsors synthesis of a new okazaki
fragment.

25. Energy from ATP is required
Melting of DNA by DnaA,
• Release of DnaB at the Forks by DnaC
⚫ Helicase action of DnaB,
• Swivel action of DNA gyrase,
⚫ Activation of primase DnaG by DnaB, and
• Activation of DNA polymerase 3 to begin
replication.

26. Elongation
In elongation step, the synthesis of two new
daughter strands takes place complementary to the
template strands.
DNA polymerases are the enzymes that synthesize
thenew daughter DNA molecules.
DNA polymerases in prokaryotes are three types:
DNA polymerase I
DNA polymerase II
DNA polymerase III

27. DNA polymerases can synthesize in one direction only which
means they can add DNA nucleotides in 5' to 3' direction.
⚫ DNA polymerases require primer to initiate the synthesis.
• Primer is 5-10 RNA nucleotide sequences that is essential
for the synthesis of new strand of DNA.
⚫ DNA polymerase can now extend this RNA primer,adding
nucleotide one by one that are complementary strand
The addition of new nucleotides require energy (ATP). This
energy is obtained from the nucleotides that have three
phosphate attached to them.

28. Leading and lagging strand
Leading strand
• Strand which runs from 5' to 3'
towards the replication fork.
• A single primer is required and
then the strand can be extended
by the action of DNA pol III.
Lagging strand
• Strand that runs from 5'to 3'
away from replication fork.
• Create fragments known as
Okazaki fragments.
• This strand requires more than
one primer.
• The polymerization is
discontinuous.

30. DNA polymerase I
Enzyme that has exonuclease activity in which it
removes RNA primer
Replace it DNA sequences.
DNA polymerase II
Enzymes that catalyzes the repair of nucleotide bas
pairs.

31. DNA polymerase III
⚫ Enzyme that synthesizes the daughter strands and also
adds nucleotides one by one to the growing DNA chain.
• Main enzyme that adds in the 5' to 3' direction.
⚫ DNA polymerase III uses 3'-hydroxyl group of the RNA
primer as an acceptor of the first DNA sequence.

33. B subunit enzyme
• It is made up of two identical protein chain to
come together to form a circle. The circle can be
loaded onto the template like a clamp to hold the
DNA pol III enzyme to the DNA.
Is referred to as sliding clamp.
⚫ Helps to hold the DNA polymerase in place when
nucleotides are being added.

34. Proffeading
DNA polymerase III check its work with each base it
adds.
⚫ Is important for the survival of an organism that
their DNA be replicated without any error.
⚫ Mismatch or error leads to mutation.
If any mismatch nucleotide is detected, it will be
removed and is replaced by accurate nucleotide.

35. DNA polymerase I
⚫ The removal and replacement of primer segments
is catalyzed by DNA pol I

36. DNA ligase
• After the action DNA pol I, nick is formed.
⚫ Nick is discontinuity in a double stranded molecule
where there is no phosphodiester bond between adjacent
nucleotides.
⚫ Nick is sealed by DNA ligase.
In lagging strand, the nick is sealed and ultimately joining
the Okazaki fragments into complete strands.

37. Termination
Replication of bacterial genome proceeds bidirectional 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.
Ter is a binding site for TUS (terminus utilization
substance)protein.

38. E.Coli DNA. Termination sites like A, B, C, D, F and G are
found to present in DNA. Of these sites, Ter A terminates
the counter clockwise moving fork while ter C terminates
the clockwise moving forks. The other sites are backup
sites

39. Tus protein binds with Ter to form a Tus-Ter complex.
Ter sequence have two binding sites permissive and Non-
permissive.

40. One complex, One
direction
⚫ Fork arrested from one
direction.
After the complete
synthesis, two duplex DNA
are found to be catenated
(knotted). This catenation
removed by the action of
topoisomerase. Finally, from
single parental duplex DNA,
two progeny duplex DNA
synthesized

42. Proposed model of DNA replication
in the late 1950s, three different mechanisms were
proposed for the replication of DNA
⚫ Conservative model
Both parental strands stay together after DNA
replication
• Semi-conservative model
The double-stranded DNA contains one parental and
one daughter strand following replication
⚫ Dispersive model Parental
and daughter DNA are interspersed in both strands
following replication
• DNA Replication in prokaryotes is semi
conservative.

43. Conclusion
DNA is very important for life.
• DNA replicates semi-conservatively.
During replication, the strands separate, replication
occurs and the two daughter DNAs are formed.
Each strands contains one parental strand and one
new strand.