DNA replication is fundamental process occurring in all living organism to copy their DNA. The process is called replication in sense that each strand of dsDNA serve as template for reproduction of complementary strand.

1. DNA Replication
Dr. M.Sankareswaran
Assistant professor, Department of Microbiology.

4. DNA REPLICATION
DNA replication is fundamental process occurring in all
living organism to copy their DNA.The process is called
replication in sense that each strand of ds DNA serve as
template for reproduction of complementary strand.

6. General feature of DNA replication
DNA replication is semi conservative
It is bidirectional process
It proceed from a specific point called origin
It proceed in 5’-3’ direction
It occur with high degree of fidelity
It is a multi-enzymatic process

9. Models of DNA replication
There were three models suggested for DNA
replication:
•Conservative,
•Semi-conservative, and
•Dispersive.

11. Conservative Method
The conservative method of replication suggests that parental
DNA remains together and newly-formed daughter strands are
also together.
Semi-Conservative Method
The semi-conservative method of replication suggests that the
two parental DNA strands serve as a template for new DNA and
after replication, each double-stranded DNA contains one strand
from the parental DNA and one new (daughter) strand.
Dispersive Method
The dispersive method of replication suggests that, after
replication, the two daughter DNAs have alternating segments of
both parental and newly-synthesized DNA interspersed on both
strands.

16. DNA replication occurs by three steps
1. Initiation:
• Initiation complex formation
• Closed complex formation
• Open complex formation
2. Elongation:
• Leading strand synthesis
• Lagging strand synthesis
3.Termination

17. Initiation
DNA replication demands a high degree of accuracy because
even a minute mistake would result in mutations.Thus,
replication cannot initiate randomly at any point in DNA.
For the replication to begin there is a particular region called
the origin of replication.This is the point where the
replication originates. Replication begins with the spotting of
this origin followed by the unwinding of the two DNA strands.
Unzipping of DNA strands in its entire length is unfeasible
due to high energy input. Hence, first, a replication fork is
created catalyzed by polymerases enzyme which is an
opening in the DNA strand.

18. Elongation
As the strands are separated, the polymerase enzymes start
synthesizing the complementary sequence in each of the
strands.The parental strands will act as a template for newly
synthesizing daughter strands.
It is to be noted that elongation is unidirectional i.e. DNA is
always polymerized only in the 5′ to 3′ direction. Therefore, in
one strand (the template 3‘→5‘) it is continuous, hence called
continuous replication while on the other strand (the
template 5‘→3‘) it is discontinuous replication.They occur as
fragments called Okazaki fragments.The enzyme called DNA
ligase joins them later.

19. Elongation
Leading strand synthesis:
•Leading strand synthesis is more a straight forward 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 proceed continuously keeping
pace with unwinding of replication fork until it encounter the
termination sequences.
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

20. REPLICATION FORK
•The replication fork is a structure that forms within the
nucleus during DNA replication.
•It is created by helicases, which break the hydrogen bonds
holding the two DNA strands together.The resulting
structure has two branching "prongs", each one made up of a
single strand of DNA.
•These two strands serve as the template for the leading and
lagging strands, which will be created as DNA polymerase
matches complementary nucleotides to the templates; the
templates may be properly referred to as the leading strand
template and the lagging strand template.

22. REPLICATION BUBBLE
•It is formed during replication in both eukaryotic and
prokaryotic DNA. It is the place where replication occurs
actively.
• It is otherwise known as replication bubble. Formation of the
replication eye provides the theta like structure to the circular
DNA during replication in prokaryotes.
•Each replication bubble found to have two replication forks,
each at the corner of an eye.

23. SINGLE-STRANDED DNA-BINDING PROTEIN
(SSB)
It was called a DNA-unwinding protein (M.W. 22,000).
Single-stranded DNA-binding protein, or SSB, binds to single-
stranded regions of DNA to prevent premature annealing, to
protect the single-stranded DNA from being digested by
nucleases, and to remove secondary structure from the DNA to
allow other enzymes to function effectively upon it

24. PRIMER
A primer is a strand of nucleic acid that serves as a starting
point for DNA synthesis. It is required for DNA replication
because the enzymes that catalyze this process, DNA
polymerases,
can only add new nucleotides to an existing strand of DNA.The
polymerase starts replication at the 3'-end of the primer, and
copies the opposite strand.
In most cases of natural DNA replication, the primer for DNA
synthesis and replication is a short strand of RNA.

25. OKAZAKI FRAGMENTS
Fragments synthesized during lagging strand formation of replication
was identified and proved by Rejis Okazaki. Hence, by his name these
fragments are called as Okazaki’s fragments.
They are short polynucleotides with 1000-2000 base pairs in length.
These fragments are synthesized by DNA polymerases.
Even though they are formed during replication, they are joined to form
larger DNA at the completion of replication by the action of DNA ligase.
The invention of Okazaki fragments lead to the proposal of
semidiscontinuous replication concept

30. ENZYMES INVOLVED IN DNA
REPLICATION

31. Enzymes Involved In DNA Replication
DNA replication is a highly enzyme-dependent process.There are many enzymes involved in
the DNA replication which includes the enzymes DNA-dependent DNA polymerase, helicase,
ligase, etc. Among them, DNA-dependent DNA polymerase is the main enzyme.
DNA-dependent DNA polymerase
It helps in the polymerization and catalyzes and regularises the whole process of DNA
replication with the support of other enzymes. Deoxyribonucleoside triphosphates are the
substrate as well as the energy provider for the replication process. DNA polymerase is of three
types:
DNA Polymerase I
It is a DNA repair enzyme. It is involved in three activities:
5′-3′ polymerase activity
5′-3′ exonuclease activity
3′-5′ exonuclease activity
DNA Polymerase II
It is responsible for primer extension and proofreading.
DNA Polymerase III
It is responsible for in vivo DNA replication.
randed DNA and protects it from forming secondary structures

32. Enzymes Involved In DNA Replication
Helicase
Helicase is the enzyme which unzips the DNA strands by breaking the hydrogen
bonds between them.Thus, it helps in the formation of the replication fork.
Ligase
Ligase is the enzyme which glues the discontinuous DNA strands.
Primase
This enzyme helps in the synthesis of RNA primer complementary to the DNA
template strand.
Endonucleases
These produce a single-stranded or a double-stranded cut in a DNA molecule.
Single-stranded Binding Proteins
It binds to single-stranded DNA and protects it from forming secondary structures

34. ENZYMES INVOLVED IN DNA REPLICATION
– DNA Helicase
– DNA Polymerase
– DNA clamp
– Single-Strand Binding (SSB) Proteins
–Topoisomerase / DNA Gyrase
– DNA Ligase
– Primase

35. DNA Helicase
• Helicases were discovered in E. coli in 1976 and are
a class of enzymes vital to all living organisms.
• Also known as helix destabilizing enzyme, they
separates the two strands of DNA at the Replication
Fork behind the topoisomerase.
•They are motor proteins that move directionally
along a nucleic acid phosphodiester backbone,
separating two annealed nucleic acid strands
(i.e., DNA,RNA, or RNA-DNA hybrid) using energy
derived from ATP hydrolysis.
•They have molecular weight 300,000, which
contain SIX identical sub units.

37. DNA GYRASE
DNA gyrase, often referred to simply as gyrase, is an
enzyme that relieves strain while double-strand DNA
is being unwound by helicase.This causes negative
supercoiling of the DNA.The ability of gyrase to relax
positive supercoils comes into play during DNA
replication.The right-handed nature of the DNA
double helix causes positive supercoils to accumulate
ahead of a translocating enzyme, in the case of DNA
replication, a DNA polymerase.The ability of gyrase
(and topoisomerase IV) to relax positive supercoils
allows superhelical tension ahead of the polymerase
to be released so that replication can continue.

39. DNA Polymerase
• DNA polymerases are enzymes that synthesize DNA molecules
from deoxyribonucleoti des, the building blocks of DNA.
•These enzymes are essential to DNA replication and usually work in
pairs to create two identical DNA strands from a single original DNA
molecule.
• During this process, DNA polymerase “reads” the existing DNA
strands to create two new strands that match the existing ones
• Also performs proof-reading and error correction.

42. DNA polymerases are enzymes that synthesize a new strand of DNA
complementary to an existing DNA or RNA template.
A polymerase enzyme, the terms ‘DNA- dependent’ or ‘RNA-
dependent’ may be used to indicate the type of nucleic acid template
that the enzyme uses:
DNA-dependent DNA polymerase copies
DNA into DNA
DNA-dependent RNA polymerase transcribes
DNA into RNA.
RNA-dependent DNA polymerase copies
RNA into DNA.

44. DNA
Polymerase
s I
• This was firstly discovered in 1958 by Arthur Kornberg who received Noble Prize in
Physiology and Medicine in 1959
• DNA Polymerases I is mainly responsible for:
• Proofreading of DNA strand
• Repairing of damage DNA
• Filling the gap between the okazaki fragments
• Removal of RNA primer

45. DNA Polymerases II
• DNA Polymerases II was identified later during the experiment on mutant E.coli
cell line
• DNA Polymerases II have temporary function when DNA Polymerases I and DNA
Polymerases III are not functional
• Still capable for doing synthesis on damage template
• Participating in DNA repairing

46. DNA Polymerases III
• DNA Polymerases III was identified later during the experiment on mutant E.coli
cell line.
• DNA Polymerases III is heterodimer enzyme composed of ten different subunits
• It is true enzyme that is responsible for the elongation process
• It also responsible for the polymerization of Nucleotide and make most of the
DNA during replication.

49. RNA Polymerase
• Enzyme that synthesizes RNA using a DNA template through a process called
transcription
• RNA polymerase enzymes are essential to life and are found in all organisms and
many viruses
• It polymerizes ribonucleotide at the 3 end of an RNA transcript

54. Single-Strand Binding (SSB) Proteins
• Single stranded binding proteins prevent
reannealing (binding of complementary DNA
sequences), protect the single-stranded DNA from
being digested by nucleases, and prevent secondary
structure formation, thereby allowing other
enzymes to function effectively on the single
strand.
• Molecular weight of the SSB protein is 75,600.
• It contains FOUR identical subunits, which binds
single stranded DNA.

56. Topoisomerase
• Every cell has enzymes that increase (or) decrease
the extent of DNA unwinding are called “Topoisomerases.
•Topoisomerase is also known as “DNA Gyrase” and
that act on the topology of DNA.
• “Topoisomerases bind to double-stranded DNA and
cut the phosphate backbone of either one or both the
DNA strands, this intermediate break allows the
DNA to be untangled or unwound, and, at the end of
these processes, the DNA backbone is resealed again.
•Topoisomerases” is an enzyme that can change the
“Linking number”(Lk).

58. •The linking number (Lk) is a topological property, it can be
defined as “ the number of times the second strand pierces the
second strand surface”.
•There are two classes of topoisomerases.
―a)Type-ITopoisomerases
―b)Type-IITopoisomerases
a)Type-ITopoisomerases:
•This act by transiently breaking one of the two DNA strands,
rotating one of the ends about the unbroken strand, and
rejoining the broken ends; they change Lk in increments of 1.
b)Type-IITopoisomerases:
•The enzyme breaks both DNA strands and change Lk in
increments of 2.

59. Topoisomer
ase

60. Class I DNA Topoisomeras

63. DNA Ligase
• DNA ligase is a specific type of enzyme, a ligase, that
facilitates the joining of DNA strands together by catalyzing the
formation of a phosphodiester bond.
• DNA ligase is used in both DNA repair and DNA replication.
•The mechanism of DNA ligase is to form two covalent
phosphodiester bonds between 3’ hydroxyl ends of one
nucleotide, ("acceptor") with the 5' phosphate end of another
("donor").
• ATP is required for the ligase reaction, which proceeds in three
steps:

64. i. Adenylation (addition of AMP) of a lysine residue in the active
center of the enzyme, pyrophosphate is released;
ii.Transfer of the AMP to the 5' phosphate of the socalled donor,
formation of a pyrophosphate bond;
iii. Formation of a phosphodiester bond between the 5'
phosphate of the donor and the 3' hydroxyl of the acceptor.

66. Primase
• DNA primase is an enzyme involved in the replication
of DNA and is a type of RNA polymerase.
• Primase catalyzes the synthesis of a short RNA (or
DNA in some organisms) segment called a primer
complementary to a ssDNA template.
• Primase is of key importance in DNA replication because no
known replicative DNA polymerases can initiate the synthesis
of a DNA strand without an initial RNA or DNA primer (for
temporary DNA elongation).
• After this elongation the RNA piece is removed by a 5'
to 3' exonuclease and refilled with DNA

70. Rolling Circle Model of
DNA Replication

71. Replication in eukaryotes is bidirectional, this type is
unidirectional.
Ideal example of this type is the circular plasmid of bacteria,
as it happens only in circular genomes

74. Initiation
Initiates
- phosphate ends, by the action of:
a) Helicase
b)Topoisomerases
c) Single stranded binding proteins (SSBPs).

75. Elongation
For Elongation, -OH group
of broken strand, using the unbroken strand as a template.
The polymerase will start to move in a circle for elongation,
due to which it is named as Rolling circle model
end will be displaced and will grow out like a waving thread.

76. 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 linear DNA
molecule.
The linear single stranded molecule is circularized by the
action of ligase and then replication to double stranded
circular plasmid molecule.