This document discusses DNA replication in prokaryotes and eukaryotes. It provides an overview of the key steps and enzymes involved in DNA replication for both prokaryotes and eukaryotes. For prokaryotes, it describes initiation at the origin of replication involving DNA A protein, elongation by DNA polymerase III, and termination when replication forks meet. For eukaryotes, it outlines initiation involving pre-replication complexes, elongation involving leading and lagging strand synthesis, and the various enzymes involved such as DNA polymerases and helicases.

1. Group Members
• Ayesha Shoaib
• Muhammad Mubashar Noor
• Sajida Batool
• Farid ullah
DNA Replication

2. Different Modes Of Replication:
Basic Rules for Replication of DNA:
• The primary role of any mode of replication is to duplicate the base sequence of
the parent molecule.
• The specificity of base pairing adenine with thymine and guanine with cytosine
provides the mechanism used by all replication systems.
• Replication of DNA occurs based on
the Chargaff’s Rule that is:
• Cytosine – Guanine ( 3- Hydrogen bonds)
• Adenine – Thymine (2- Hydrogen bonds)

3. • Nucleotide monomers are added one by one to the end of a growing strand by
an enzyme called DNA polymerase.
• The sequence of bases in each new or daughter strand is complementary to the
base sequence in the original template or parent strand being copied that is, if
there is an adenine in the parent strand, a thymine nucleotide will be added to
the end of the growing daughter strand when the adenine is being copied.

4. Semiconservative Process of DNA Replication
• When Watson and Crick proposed the double helical structure of DNA with its
complementary base pairing, they immediately recognized that base pairing
specificity could provide the basis for duplication.
• If the two complementary strands of a double helix separated, (by breaking the
Hydrogen bond) each parental strand could direct the synthesis of a new
complementary strand.
• That is each parental strand could serve as a template for a new complementary
strand.
• If Adenine is in parent strand then synthesis of Thymine in complementary strand
will take place.
• This mechanism of DNA replication is called semiconservative replication.
• In considering possible mechanism of DNA replication, three different
hypothetical modes are apparent.

5. Modes of DNA Replication:
• The three models for DNA replication:

6. • Conservative Model:
In the conservative model, the parental molecule directs synthesis of an entirely new
double-stranded molecule, such that after one round of replication, one molecule is
conserved as two old strands. This is repeated in the second round.
• Dispersive Model:
In the dispersive model, material in the two parental strands is distributed more or
less randomly between two daughter molecules.

7. • Semi-conservative Model:
In the semi-conservative model, the two parental strands separate and each
makes a copy of itself. After one round of replication, the two daughter molecules
each comprises one old and one new strand.
After two rounds, two of the DNA molecules consist only of new material, while the
other two contain one old and one new strand.
A classic experiment by Meselson and Stahl provided evidence that DNA replicates
semiconservatively.

8. Semi-conservative Mode Of Replication – Experiment By
Meselson And Stahl At California Institute Of Technology In
1958:

9. The Meselson-stahl Experiment, Which Showed That DNA
Replicates Semi-conservatively:

10. DNA Replication Is Semi- Conservative:
• Experimental Proof Matthew Meselson and Franklin Stahl .
Conclusion:
• Semi-conservative model is the most appropriate for replication.

11. Rolling Circle Mechanism:
• Rolling circle replication is a process of unidirectional nucleic acid
replication.
• It can rapidly synthesize multiple copies of circular molecules of RNA or
DNA, such as plasmid.
• Widely used in molecular biology & biochemical nanotechnology,
especially in the field of biosensing.
• Applies to replication of several viral DNAs such as λ-phage,M-13 phage.
• Circular DNA in mitochondria and chloroplast also replicate through this
mechanism.
• E.g.: Replication of E-coli Factor-F during conjugation ,for transfer of DNA
from donor to recipient.

12. Three steps of Rolling Circle Replication :
(1) Initiation
• In the process of initiation, three enzymes works together such as:
Helicase
Topoisomerase
Single stranded binding proteins (SSBPs)
• These enzymes bind with phosphate ends of nick strand.

13. (2) Elongation
• In the process of elongation, 3’OH group of broken strand, work as a template.
The polymerase enzyme help to move in a circle for elongation, due to which it is
named as rolling circle mechanism. 5’ end will be displaced and grow like as a
thread.

14. (3)Termination
• At the point of termination, the linear DNA molecule is cleaved from the circle
resulting in a double stranded circular DNA molecule and a single stranded DNA
molecule(ssDNA).
• The linear single stranded molecule is circularized by the action of ligase and then
replication to double stranded circular plasmid molecule.

15. Various steps involve in this process:
• Firstly, Initiator protein Rep A bind to nicked 5’ OH end of ssDNA at ori site.
• After that DNA Polymerase enzyme bind with 3’P end of double stranded
(dsDNA)or ori site.

16. • Then helicase enzyme attach with RepA protein and start replication process. In
this process 5’ end of ssDNA elongated with the help of single stranded binding
protein.
• After completing new ssDNA synthesis, new strand separate with the single
stranded binding protein from the nicked region and nick place sealed by DNA
ligase enzyme

17. • At last single stranded DNA also synthesize its complementary strands

18. Example:
Viral DNA: Some DNA viruses replicate their genomic information in host cells via
rolling circle replication.
• Human herpes virus
• Human papilloma virus
• Gemini virus
Viral RNA: Some RNA viruses and viroids also replicate their genome through
rolling circle RNA replication.
• Pospiviridiae family
• Avsunviridiae family

19. Theta Model Of Replication
• Originally discovered by John Cairns, it led to the understanding that
bidirectional DNA replication could take place.
• A Theta structure is an intermediate structure formed during the
replication of a circular DNA molecule (prokaryotic DNA).
• Two replication forks can proceed independently around the DNA
ring and when viewed from above the structure resembles the Greek
letter "theta" (θ).

20. John Cairns Experiment
• In 1963, John Cairns performed experiment by using autoradiography.

21. Detailed Description Of The Theta Mode Of DNA Replication.

22. Examples:

23. Muhammad Mubashar Noor
04051813035
DNA Replication in
Prokaryotes

24. DNA Replication
• A process in which daughter DNA is synthesized by using
the parental DNA as template.
• Transferring the genetic information to the descendent
generation.

25. DNA Replication in Prokaryotes
• DNA Replication in prokaryotes is Semi Conservative.
Each strand of template DNA is being copied.
• DNA Replication is Semi Discontinuous.
The leading strand copies continuously
The lagging strand copies in segments (Okazaki fragments).
• DNA Replication is Bidirectional.
Both in clockwise direction and in anticlockwise direction.

27. DNA Replication
DNA Replication in prokaryotes include
• Initiation
Replication begins at the origin of replication.
• Elongation
New strands of DNA are synthesized by DNA Polymerase.
• Termination
End of replication take place.

28. Initiation
• The replication begins at a specific initiation point called Ori C site.
• The Ori C site consists of 245 base pairs, of which three of 13 base
pairs sequence are highly conserve in many bacteria and forms the
consensus sequences (GATCTNTTNTTTT) .
• Close to Ori C site, there are four of 9 base pair sequences each
(TTATCCACA)

29. DNA A Protein
• It recognizes and binds up to four 9 bp repeats in Ori C to form a complex
of negatively supercoiled Ori C. DNA wrapped around a central core of
DNA A protein monomers. This process also requires the presence of
histones.
• Once the four bp repeats are occupied 20-40 additional DNA A monomers
bind so that entire Ori C region is complexes with DNA A protein.
• The resulting complex resembles a nucleosome with negatively
supercoiled Ori C DNA wrapped around a DNA core.

30. • DNA A protein subunits then successively melt three 13 bp sequences in the
presence of ATP, which results in the formation of 45 bp open complex.
• The DNA A protein then guides a DNA B and DNA C complexes into the
melted region to form a prepriming complex.
• The DNA C is subsequently released.
• DNA B further unwinds open complex to form a prepriming complex.
• DNA gyrase , Single stranded binding proteins are bound to prepriming
complex and now the complex is called priming complex.
• DNA G protein form a primer .
• To the above complex DNA Polymerase III will bind and form a replisome.

31. Replisome
• It is the multiprotein structure
that assembles at the
bacterial replicating fork to
undertake the synthesis of
new DNA. It contains a DNA
Polymerase and other
enzymes.

32. Elongation
• In this phase the synthesis of new daughter strands take place
complementary to the template strands.
• DNA Polymerase III is the enzyme that synthesize the daughter
strands.
• 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.
• Leading strand is synthesized continuously in 5 to 3 direction along
the direction of the movement of replication fork.

33. • Lagging strand synthesis occurs discontinuously by loop formation in
short segments called Okazaki fragments.
• The lagging strand is looped so that the DNA synthesis proceeds steadily
on both the leading and lagging strand at the same time.

34. • DNA Polymerase III synthesizes the new DNA strands by adding 5-P of
deoxyribonucleotide to 3-OH group of the already present fragement.
• Thus the chain grows in 5 to 3 direction. The reaction catalyzed by DNA
Polymerase III is very fast that it can add 1000 nucleotides per second.
• The RNA primer is degraded by RNAase H enzyme and DNA Poymearse I
add actual DNA nucleotides.
• The remaining nick is sealed by DNA ligase. DNA ligase catalyses the
formation of phosphodiester bond.

35. Termination
• Termination of DNA replication occurs when two replication forks
meet on the same stretch of DNA.
• Replication terminates at the terminus region containing multiple 20
base pair sequences called as Ter sequences.
• Ter sequences works as a binding site for protein Tus(Terminus
utilization substance). It stops the DNA B protein resulting in the
termination of DNA replication.
• The completed chromosome then partitioned into two daughter cells
during cell division.

37. Sajida Batool
04051813034
DNA Replication in
Eukaryotes

38. Eukaryotic DNA Replication
 DNA replication is the process by which an organism duplicates its DNA
into another copy that is passed on to daughter cells.
 Replication occurs before a cell divides to ensure that both cells receive an
exact copy of the parent’s genetic material.
 Replication is bi-directional and originates at multiple origins of replication
(Ori C) in eukaryotes.
 DNA replication uses a semi-conservative method that results in a double-
stranded DNA with one parental strand and a new daughter strand.
 It occurs only in the S phase and at many chromosomal origins.

39. Takes place in the cell nucleus
Synthesis occurs only in the 5′ to 3′direction.
Individual strands of DNA are manufactured in different directions, producing a leading and
a lagging strand.
Lagging strands are created by the production of small DNA fragments called Okazaki
fragments that are eventually joined together.
• THE ENZYMES OF DNA REPLICATION:
• Several enzymes plays their role in the replication of DNA.
• Helicases:
• Unwind the DNA helix at the start of replication.
• SSB proteins:
• Bind to the single strands of unwound DNA to prevent reformation of the DNA helix during
replication

40. DNA Polymerases:
• Eukaryotic cells contain five different DNA polymerases; α, β, γ, δ and ε.
• DNA polymerases α and δ replicate chromosomal DNA, DNA polymerases β and
ε repair DNA, and DNA polymerase γ replicates mitochondrial DNA.
• DNA polymerase α and δ synthesize the lagging strand, via Okazaki fragments.
• The RNA primers are synthesized by DNA polymerase α which carries a primase
subunit.
• DNA polymerase δ synthesizes the leading strand.
• Telomerase:
• A DNA polymerase that contains an integral RNA that acts as its own primer, is
used to replicate DNA at the ends of chromosomes (telomeres).
• DNA topoisomerase :
• The overall function of DNA topoisomerase is to manage the topological state of
the DNA in the cell

41. Histone Dissociation and Association
Since DNA is present in packaged form as chromatin, DNA replication is
sandwiched between two additional steps in eukaryotes.
 Dissociation of histone:
Methylation at the fifth position of cytosine residues by a DNA methyl
transferase appears to functioning by loosening up the chromatin structure.
 Synthesis of histone:
The synthesis of new histone occurs simultaneously with DNA replication.

42. Pre replication complex
• Pre replication complex forms at the origion of replication during G1phase.
• In most eukaryotes it is composed of ORC, Cdc6 ,Cdt1 and MCM.
• ORC stands for origin replication complex.
• It recognizes and binds with origion of replication.
• Origin DNA sequences usually have many adenine–thymine (AT) base pairs and
are said to be “AT rich.”
• This makes sense because less energy is required to melt the two hydrogen bonds
joining A with T, compared with the three hydrogen bonds joining guanine–
cytosine (GC) base pairs.
• All the other proteins Cdc6 ,Cdt1 and MCM bind with ORC and this whole
complex is known as, pre ,initiation complex.
• First of all Cdc6 know as cell division cycle 6 binds with ORC.
• After that Cdt1 which is chromatin licensing DNA replication factor 1 helps
minichromosome maintenance protein complex to bind with ORC.

43. Initiation complex
• The main purpose of Cdc6 and Cdt1 is to load MCM on DNA.
• After loading of mcm these proteins are phosphorylated for the
activation of initiation complex.
• This complex is formed during the transition of G1 phase to S phase.
• Cyclic dependent kinase CDK and Dbf4 proteins kinase phosphorylate
Cdc6 and Cdt1 make this complex active for unwinding of DNA.

44. Activator complex
• After the activation of initiation complex two more complex factors Cdc45 and GIN
activates the the MCM helicase activity.
• Therefore, this complex is known as activator complex which is formed during the
S phase of cell cycle.
• It unwinds the DNA and replication fork is formed.

45. Elongation
• Once the initiation complex is formed and the cells pass into the S phase, the
complex then becomes a replisome and elongation is initiated.
• Once the elongation is initiated, it form the replication fork by unwinding the
DNA strand.
• As the double helix of DNA separates from one side and super coils are formed on
the other side.
• The problem of super coils comes in the way of DNA replication is solved by a
group of enzymes called DNA topoisomerase.

46. Leading Strand
• Leading strand synthesis is initiated upon RNA primer, synthesized by the
primase subunit of DNA pol α.
• The RNA primer contains 10-15 nucleotides.
• Then DNA pol α adds a stretch of DNA to the primer.
• At this point replication factor C (RFC) carries out a process called
polymerase switching.
• RFC removes DNA pol α and assembles PCNA in the region of primer strand
terminus.
• Then DNA pol epsilon binds to PCNA and carries out highly processive
leading strand synthesis due to its 5’- 3’ polymerization activity.
• After the addition of several nucleotides in the daughter strand, primer is
removed. DNA pol Є due to its 5’-3’ exonuclease activity removes the primer
,and the gap is filled by the same DNA pol Є due to its 5’-3’ polymerization
activity.
• Then the nick is sealed by DNA ligase
• DNA pol δ improves the fidelity of replication due to its , proof reading
activity.

47. Lagging Strand
• Lagging strand synthesis: Lagging strand synthesis of Okazaki fragment initiated same
way as leading strand synthesis.
• An Okazaki fragment contains 150-200 nucleotides.
• RNA primer is synthesized by DNA pol α due to its primase activity.
• The primer is then extended by DNA pol delta due to its 5’-3’ polymerization activity
(lagging strand synthesis), using deoxy ribonucleotides (dNTPs).
• Priming is a frequent event in lagging strand synthesis with RNA primers placed every
50 or 80 nucleotides.
• All but one of the ribonucleotides in RNA primer is removed by RNase H1. Then
exonuclease activity of FEN 1/ RTH 1 complex removes the one remaining nucleotide.
• The gap is filled by DNA pol Є by its 5’-3’ polymerase activity.
DNA ligase joins the Okazaki fragment of the growing DNA strand.

48. TERMINATION
• When the replication forks meet each other, then termination occurs.
• It will result in the formation of two duplex DNA.
• Even though replication terminated, 5’ end of telomeric part of the newly synthesized
DNA found to have shorter DNA strand than the template parent strand.
• This shortage corrected by the action of telomerase enzyme and then only the actual
replication completed.

50. TELOMERES
• Eukaryotic chromosomes are linear. The ends of chromosomes have specialized
structures known as ‘Telomeres’.
• Telomeres are short (5-8 bp) tandem repeated and GC rich nucleotide sequence.
Telomeres form protective cap 7-12 kbp long in the ends of chromosome.
• Telomeres are necessary for chromosome maintenance and stability. They are
responsible for maintaining chromosome integrity by protecting against DNA
degradation and rearrangement.

51. Problem in the completion of replication of lagging strand::
The limitations of DNA polymerase create problems for the linear DNA of eukaryotic
chromosomes.
When a linear DNA molecule replicates, a gap is left at the 5’ end of each new strand
because DNA polymerase can only add nucleotides to a 3’ end.
As a result, with each round of replication, the DNA molecules get slightly shorter
because the usual replication machinery provides no way to complete the 5 ends of
daughter strands, so repeated rounds of replication produce shorter DNA molecules.
Even if an Okazaki fragment can be started with an RNA primer bound to the very end
of the strand, once that primer is removed, it cannot be replaced with DNA because there
is no 3’ end available for nucleotide addition.

53. Telomerase:
• Telomerase is ribonucleoprotein.
• It contains RNA component which has repeat of 9 to 30 nucleotides long.
• This RNA component serves as the template for the synthesis of telomeric repeats
at the parental DNA ends.
• Telomerase is RNA dependent DNA polymerase with a RNA component.
• Telomerase uses the 3’ end of parental DNA strand as primer, RNA component of
telomerase as template, adds successive telomeric repeats to the parental DNA
strand at its 3’ end due to its 5’-3’ RNA dependent DNA polymerase activity

55. Faridullah
04051813011
Enzymology and Differences between
Prokaryotic and Eukaryotic Replication

56. Prokaryotes DNA replication :Enzymes and their
function
1) DNA Polymerase 1 :
Primary required for repair.
2) DNA Polymerase 2 :
Primary required for repair.
3) DNA Polymerase 3 :
It is the enzymes required DNA synthesis.
4) DNA Helicase :
It is the enzymes which unwinds the DNA by breaking the DNA bonds.

57. Prokaryotes DNA replication :Enzymes and their
function
5) Ligase :
Seals the gap between okazai fragments to create one continuous
strand.
6) Primase :
• Synthesizes RNA primers needed to start replication.
7) Sliding clamp:
• It keeps DNA polymerase attached to the template while the
polymerase synthesis a new strand.

58. Prokaryotes DNA replication :Enzymes and
their function
8) Topoisomerase:
• Helps relive to stress on DNA when unwinding by causing breaks and
then resealing the DNA .
9) Single strain binding protein :
• Bind to single stranded DNA to avoid DNA rewinding back.

59. Eukaryotes DNA replication :Enzymes and
their function
1)Helicase
• It is the enzymes which unwinds the DNA by breaking the DNA
bonds.
2) Single strain binding protein :
• Bind to single stranded DNA to avoid DNA rewinding back.
3)Primase:
• Synthesis of RNA primer.

60. Eukaryotes DNA replication :Enzymes and
their function
4)DNA Polymerase Delta :
• Synthesis of DNA of leading strand.
5)DNA Polymerase Alpha:
• Synthesis of DNA of lagging strand.
6)DNA Ligase:
• Joining of okazaki fragments.
7)DNA Topoisomerase 2:
• Removal of positive supercoils ahead of advancing replication fork.

61. Property Prokaryotes Eukaryotes
Origin of Replication Single Multiple
Rate of replication 1000 nucleotide/s 50 to 100nucleotide/s
DNA polymerase type 5 14
Telomerase Not present Present
Strand elongation DNA pol 3 Pol Alpha,Pol Delta,Pol Epsolon
Differences between Prokaryotic and
Eukaryotic DNA Replication