The process by which DNA molecule makes its identical copies is known as DNA replication or DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule

1. Dr. Pawan Kumar Kanaujia
Assistant Professor
Molecular Biology
(Theory)
DNA Replication

2. The process by which DNA molecule makes its identical copies is known as
DNA replication or DNA replication is the biological process of producing
two identical replicas of DNA from one original DNA molecule. It takes
place in S-phase of interphase.
Replication patterns are somewhat different in procaryotes and eucaryotes.
In procaryotes
 when the circular DNA chromosome of E. coli is copied, replication
begins at a single point, the origin.
Synthesis occurs at the replication fork, the place at which the DNA
helix is unwound and individual strands are replicated.
Two replication forks move outward from the origin until they have
copied the whole replicon, that portion of the genome that contains
an origin and is replicated as a unit.
When the replication forks move around the circle, a structure shaped
like the Greek letter theta (θ) is formed.
Finally, since the bacterial chromosome is a single replicon, the forks
meet on the other side and two separate chromosomes are released.

3. In eukaryotes
Eucaryotic DNA is linear and much longer than procaryotic DNA;
E. coli DNA is about 1,300 µm in length, whereas the 46
chromosomes in the human nucleus have a total length of 1.8 m
(almost 1,400 times longer).
Clearly many replication forks must copy eucaryotic DNA
simultaneously so that the molecule/DNA can be duplicated in a
relatively short period, and so many replicons are present that there
is an origin about every 10 to 100 µm along the DNA.
Replication forks move outward from these sites and eventually
meet forks that have been copying the adjacent DNA stretch. In this
fashion a large molecule is copied quickly.

4. FIGURE 10.12 Mechanism of action of DNA
polymerases: covalent extension of a DNA
primer strand in the 5’ → 3’ direction. The
existing chain terminates at the 3 end with
the nucleotide deoxyguanylate
(deoxyguanosine-5-phosphate). The
diagram shows the DNA polymerase-
catalyzed addition of deoxythymidine
monophosphate (from the precursor
deoxythymidine triphosphate, dTTP) to
the 3’end of the chain with the release of
pyrophosphate (P2O7).

5. Initiation, elongation and termination are three main steps in DNA
replication. Let us now look into more detail of each of them:
Step 1: Initiation
The point at which the replication begins is known as the Origin of
Replication (oriC). Helicase brings about the procedure of strand
separation, which leads to the formation of the replication fork.
Step 2: Elongation
The enzyme DNA Polymerase III makes the new strand by reading the
nucleotides on the template strand and specifically adding one
nucleotide after the other. If it reads an Adenine (A) on the template,
it will only add a Thymine (T).
Step 3: Termination
When Polymerase III is adding nucleotides to the lagging strand and
creating Okazaki fragments, it at times leaves a gap or two between
the fragments. These gaps are filled by ligase. It also closes nicks in
double-stranded DNA.

8. Process of Replication
During replication Each enzyme and protein have their own
specific function.
Let us look at the process step by step

9. Initiation
Helicase – The point at which the replication begins is known as the
Origin of Replication. Helicase brings about the procedure of strand
separation, which leads to the formation of the replication fork.
It breaks the hydrogen bond between the base pairs to separate the
strand. It uses energy obtained from ATP Hydrolysis to perform the
function.
SSB Protein – Next step is for the Single-Stranded DNA Binding
Protein to bind to the single-stranded DNA. Its job is to stop the
strands from binding again.
DNA Primase – Once the strands are separated and ready, replication
can be initiated. For this, a primer is required to bind at the Origin.
Primers are short sequences of RNA, around 10 nucleotides in length.
Primase synthesizes the primers.

10. Elongation
DNA Polymerase III – This enzyme makes the new strand by reading
the nucleotides on the template strand and specifically adding one
nucleotide after the other. If it reads an Adenine (A) on the template, it
will only add a Thymine (T). It can only synthesize new strands in the
direction of 5’ to 3’. It also helps in proofreading and repairing the new
strand. Now you might think why does Polymerase keep working along
the strand and not randomly float away? Its because a ring-shaped
protein called as sliding clamp holds the polymerase into position.

11. Now when replication fork moves ahead and the Polymerase III starts
to synthesize the new strand a small problem arises. If you remember,
I mentioned that the two strands run in the opposite directions. This
means that when both strands are being synthesized in 5’ to 3’ direction,
one will be moving in the direction of the replication fork while the
other will move in the opposite.
The strand, which is synthesized in the same direction as the replication
fork, is known as the ‘leading’ strand. The template for this strand runs
in the direction of 3’ to 5’. The Polymerase has to attach only once and
it can continue its work as the replication fork moves forward. However,
for the strand being synthesized in the other direction, which is known
as the ‘lagging’ strand, the polymerase has to synthesize one fragment
of DNA. Then as the replication fork moves ahead, it has to come and
reattach to the new DNA available and then create the next fragment.
These fragments are known as Okazaki fragments (named after the
scientist Reiji Okazaki who discovered them).

12. Termination
DNA Polymerase I – If you remember, we had added a RNA primer at
the Origin to help Polymerase initiate the process. Now as the strand
has been made, we need to remove the primer. This is when
Polymerase I comes into the picture. It takes the help of RNase H
to remove the primer and fill in the gaps.
DNA ligase – When Polymerase III is adding nucleotides to the lagging
strand and creating Okazaki fragments, it at times leaves a gap or two
between the fragments. These gaps are filled by ligase. It also closes
nicks in double-stranded DNA.
The Replication process is finally complete once all the primers are
removed and Ligase has filled in all the remaining gaps. This process
gives us two identical sets of genes, which will then be passed on to
two daughter cells. Every cell completes the entire process in just one
hour! The reason for taking such short amount of time is multiple
Origins. The cell initiates the process from a number of points and then
the pieces are joined together to create the entire genome!`

13. Mechanism of DNA Replication:
Mechanism of DNA replication is the direct result of DNA double helical
Structure proposed by Watson and Crick. It is a complex multistep
process involving many enzymes.
Initiation:
It involves the origin of replication. Before the DNA synthesis begins,
both the parental strands must unwind and separate permanently into
single stranded state. The synthesis of new daughter strands is initiated
at the replication fork. In fact, there are many start sites.
Elongation:
The next step involves the addition of new complementary strands.
The choice of nucleotides to be added in the new strand is dictated by the
sequence of bases on the template strand. New nucleotides are added one by
one to the end of growing strand by an enzyme called DNA polymerase.
There are four nucleotides, deoxyribrnucleotide triphosphates dGTP, dCTP,
dATP, dTTP present in the cytoplasm.
Termination:
All the end termination reactions occur. Duplicated DNA molecules are
separated from one another. The purpose of DNA replication is to create
two daughter DNA molecules which are identical to the parent molecule.

14. Replication: 1st step
 Unwind DNA
 helicase enzyme
 unwinds part of DNA helix
 stabilized by single-stranded binding proteins
 PREVENTS DNA MOLECULE FROM CLOSING!
 DNA gyrase
 Enzyme that prevents tangling upstream from the replication
fork
single-stranded binding proteins replication fork
helicase gyrase

15. Replication: 2nd step
 RNA Primase
 Adds small section of RNA (RNA primer) to the
3’ end of template DNA
 Why must this be done?
 DNA polymerase 3 (enzyme that builds new DNA
strand) can only add nucleotides to existing strands
of DNA

16. DNA
Polymerase III
Replication: 3rd step
 Build daughter DNA
strand
 add new
complementary bases
 With the help of the
enzyme DNA
polymerase III

17. Replication: 4th step
 Replacement of RNA primer by DNA
 Done by DNA polymerase I

18. DNA polymerase III
RNA primer is added
 built by primase
 serves as starter sequence for DNA polymerase III
HOWEVER short segments called Okazaki fragments
are made because it can only go in a 5 3 direction
DNA replication on the lagging strand
5
5
5
3
3
3
5
3
5
3 5 3
growing
replication fork
primase
RNA

19. • Conservative replication model
• Dispersive replication model
• Semiconservative replication
Proposed DNA Replication Models

20. Figure 12.1 Three proposed models of replication are conservative replication,
dispersive replication, and semiconservative replication.

21. Initially, three models were proposed for DNA replication.
In conservative replication (Figure 12.1a), the entire double-stranded
DNA molecule serves as a template for a whole new molecule of DNA,
and the original DNA molecule is fully conserved during replication.
In dispersive replication (Figure 12.1b), both nucleotide strands break
down (disperse) into fragments, which serve as templates for the
synthesis of new DNA fragments, and then somehow reassemble into
two complete DNA molecules. In this model, each resulting DNA
molecule is interspersed with fragments of old and new DNA;
none of the original molecule is conserved.
Semiconservative replication (Figure 12.1c) is intermediate between
these two models; the two nucleotide strands unwind and each serves
as a template for a new DNA molecule.

22. • Two isotopes of nitrogen:
• 14N common form; 15N rare heavy form
• E. coli were grown in a 15N media first, then
transferred to 14N media.
• Cultured E. coli were subjected to equilibrium
density gradient centrifugation.
Meselson and Stahl’s Experiment

24. Bacterial DNA Replication
• Initiation: 245 bp in the oriC (single origin replicon);
an initiation protein

25. In prokaryotic chromosome contain a single “Orgin of replication” called oriC.
oriC is 245bp long and contain two different conserved repeat sequences of
13bp (AT-rich)sequence is present as three tandem repeats and another
conserved component of 9bp sequence that is repeated four times and
interspersed with other sequences. these four sequences are binding sites for
a protein that play a key role in the formation of the replication bubble.

26. • Unwinding of DNA is performed by Helicase. Gyrase removes
supercoiling ahead of the replication fork. Single stranded DNA is
prevented from annealing by single stranded binding proteins.
• Primers: an existing group of RNA nucleotides with a 3′-OH
group to which a new nucleotide can be added; usually 10 ~ 12
nucleotides long
Primase: RNA polymerase
One particular protein called Dna A is responsible for the recognition of Orgin of
replication. DnaA protein bind with 9mer, this step facilitates the subsequent
binding of helicase (DnaB & DnaC)

27. Bacterial DNA Replication
• Elongation: carried out by DNA polymerase III
• Removing RNA primer: DNA polymerase I
• DNA ligase: connecting nicks after RNA primers are
removed
• Termination: when a replication fork meets or by
termination protein

28. Figure Summary of DNA synthesis at a single replication fork. Various
enzymes and proteins essential to the process are shown.

29. Table

30. Table

31. The Taylor–Woods–Hughes experiment,
demonstrating the semi-conservative mode of
replication of DNA in root tips of Vicia faba.

32. A portion of the plant is shown in the top photograph.
(a) An unlabeled chromosome proceeds through the cell
cycle in the presence of 3H-thymidine. As it enters mitosis, both sister
chromatids of the chromosome are labeled, as shown, by
autoradiography.

33. (b), this time in the absence of 3H-thymidine, only one chromatid of
each chromosome is expected to be surrounded by grains. Except
where a reciprocal exchange has occurred between sister chromatids
(c), the expectation was upheld. The micrographs are of the actual
autoradiograms obtained in the experiment.

34. Eukaryotic DNA replication
1957, J. Herbert Taylor, Philip Woods, and Walter Hughes presented evidence that
Semiconservative Replication also occurs in Eukaryotic organism. They were used
root-tip cells of the broad bean, Vicia faba, researchers were able to monitor the
process of replication by lebeling DNA with 3H-thymidine, a radioactive precursor
of DNA, and by performing autoradiography.
Chromosomal DNA replication occurs only during the S phase of the cell cycle.
In eukaryotes, chromosome contain multiple replication origins are called
autonomously Replicating sequences (ARSs)
ARSs consist 120 bp containing a consensus sequence of AT rich 11bp . Helicase
loading required four separate proteins to act at each replicator.
 Replication origins are recognize by a six-protein complex known as an Origin
Recognisition complex (ORC), which tags the orgin as a site of initiation.
The first step in helicase loading is the recognition of the replicator by the
eukaryotic initiator, ORC, bound to ATP. As cells enter the G1 phase of the cell cycle,
ORC bound to the origin recruits two helicase loading proteins (Cdc6 and Cdt1) and
two copies of the Mcm2-7 helicase to the origin.

35. 9-30 F I G U R E. Eukaryotic helicase load
ing. Loading of the eukaryotic replicative
DNA helicase is an ordered process that is
ini tiated by the association of the ATP-bound
origin recognition complex (ORC) with the
replicator. Once bound to the replicator,
ORC recruits ATP-bound Cdc6 and two
copies of the Mcm2-7 helicase bound to
a second helicase loading protein, Cdt1.
This assembly of proteins triggers ATP
hydrolysis by Cdc6, resulting in the loading
of a head-to-head dimer of the Mcm2-7 com
plex encircling double-stranded origin DNA
and the release of Cdc6 and Cdt1 from the
origin. Subsequent ATP hydrolysis by ORC
is required to reset the process (illustrated as
release from Mcm2-7). Exchange of ATP for
ADP allows a new round of helicase loading.

36. ATP hydrolysis by Cdc6 results in the loading of a head-to-head dimer of the
Mcm2-7 complex such that they encircle the double-stranded origin DNA. During
this event, Cdt1 and Cdc6 are released from the origin.
Helicases that are loaded during G1 are only activated to unwind DNA and initiate
replication after cells pass from the G1 to the S phase of the cell cycle.
Loaded helicases are activated by two protein kinases: CDK (cyclin dependent
kinase) and DDK (Dbf4-dependent kinase).
DDK targets the loaded helicase, and CDK targets two other replication proteins.
Phosphorylation of these proteins results in the Cdc45 and GINS proteins binding
to the Mcm2-7 helicase (Fig 9-31). Importantly, Cdc45 and GINS strongly stimulate
the Mcm2-7 ATPase and helicase activities and together form the Cdc45–Mcm2-7–
GINS (CMG) complex, which is the active form of the Mcm2-7DNA helicase.
Helicase is initially loaded around dsDNA as a head-to-head dimer, at the
replication fork it is thought to act as a single Mcm2-7 hexamer encircling ssDNA.
Its mean one strand of DNA must be ejected from the central channel of each
helicase and start unwinding (the interactions between the two Mcm2-7 complexes
must be disrupted) Fig 9-32.

37. 9-31 F I G U R E. Activation of loaded helicases
leads to the assembly of the eukaryotic
replisome. As cells enter into the S phase of the
cell cycle, two kinases, CDK and DDK, are
activated. DDK phosphorylates loaded Mcm2-7
helicase, and CDK phosphorylates Sld2 and
Sld3. Phosphorylated Sld2 and Sld3 bind to
Dpb11, and together these proteins facilitate
binding of the helicase-activating proteins,
Cdc45 and GINS, to the helicase. Cdc45 and
GINS form a stable complex with the Mcm2-7
helicase (called the Cdc45/ Mcm2-7/GINS, or
CMG, complex) and dramatically activate
Mcm2-7 helicase activity. The leading-strand
DNA polymerase (Ɛ) is recruited to the helicase
at this stage (before DNA unwinding). After
formation of the CMG complex, Sld2, Sld3, and
Dpb11 are released from the origin. DNA Pol
α/primase and DNA Pol δ(which primarily act
on the lagging strand) are only recruited after
DNA unwinding. The protein–protein
interactions that hold the DNA polymerase at
the replication fork remain poorly understood.

38. F I G U R E 9-32 Helicase activation alters helicase interactions. Before
helicase activation, loaded helicases encircle double-stranded DNA and are
in the form of a head-to-head double hexamer (mediated by interactions
between the Mcm2-7 amino termini). After helicase activation, the Mcm2-7
protein in the CMG complex is proposed to encircle single-stranded DNA,
and the interaction between the two Mcm2-7 complexes has been broken.

39. F I G U R E 9-34 Cell cycle regulation of CDK
activity controls replication. In S. cerevisiae
cells, CDK levels tightly regulate helicase
loading and activation. DuringG1,CDK
levels are low, allowing helicases to be
loaded, but the loaded helicases cannot be
activated (because of the requirement of
CDK for this event). During S phase, elevated
CDK activity inhibits new helicase loading and
activates previously loaded helicases.
When a loaded helicase is used for the
initiation of replication, it is incorporated into
the replication fork and leaves the origin.
Similarly, passive replication of origin DNA
also removes the helicase from the origin
DNA (not shown). Because CDK levels remain
high until the end of mitosis, no new helicase
loading can occur until chromosome
segregation is complete and the daughter
cells have returned to G1.Without
a new round of helicase loading, reinitiation
is impossible.

40. Unwound single strands are kept in the extended state by a replication
protein A (Rp-A) (Lewin's Genes X)
Topoisomerase released torsonal strain which is develop on the head of
replication fork.
The three eukaryotic DNA polymerases α, δ & Ɛ are the major form of enzyme
involved in initiation and elongation of DNA synthesis.
DNA polymerases α/primase synthesize RNA primer of length of ~10 ribo-
nucleotides at both strands then pol αdissociates from the strands abd is replaced
by pol δ & Ɛ.
The event polymerase switching occurs, pol Ɛ synthesizes DNA on leading strand
and pol δ synthesizes DNA on laging strand and these enzymes (pol Ɛ & pol δ)
exhibits 3` to 5` exonuclease activity , thus having the potential to proofreading.
However , they do not have 5`to 3` exonuclease activity . Thus they can not remove
RNA primers.
pol Ɛ & δ must interact with PCNA (Proliferating Cell Nuclear Antigen) and
replication factor (Rf-C). PCNA is a trimeric protein that forms a closed ring; Rf-C
induces a change in the conformation of PCNA that allow it to encircle DNA, providing
the essential sliding clamp to prevent the polymerase from falling off the strands.

41. The RNA primer are excised by RNase H or two nucleases ribonuclease H1 (which
degrades RNA present in RNA-DNA duplexes) & ribonuclease FEN-1 (F1 nuclease1).
pol Ɛ & δ fill the gap at lagging and leading strands respectively, except a nicks or
break in the phosphodiester back bone between the 3’-OH & 5’ phosphate. This
nick in the new DNA strands repaired by an enzyme called DNA ligase.
A number of proteins are involved in the disassembly and assembly of
nucleosomes during chromosome replication in eukaryotes. Two of the most
important are nucleosome assembly protein-1 (Nap-1) and chromatin assembly
factor-1 (CAF-1).
Nap-1 transports histones from their site of synthesis in cytoplasm to the nucleus
& CAF-1 delivers histones to the site of DNA replication by binding to PNCA clamp
that lead (tethers) to DNA polymerase Ɛ & δ to DNA template.

42. FIGURE 10.32

43. FIGURE 10.32 Some of the important components of a replisome in eukaryotes.
Each replisome contains three different polymerases, α, δ, and Ɛ. The DNA
polymerase α-DNA primase complex synthesizes the RNA primers and adds short
segments of DNA. DNA polymerase δ then completes the synthesis of the Okazaki
fragments in the lagging strand, and polymerase Ɛ catalyzes the continuous
synthesis of the leading strand. PCNA (proliferating cell nuclear antigen) is
equivalent to the β subunit of E. coli DNA polymerase III; it clamps polymerases δ
and Ɛ to the DNA molecule facilitating the synthesis of long DNA chains.
Ribonucleases H1 and FEN-1 (F1 nuclease 1) remove the RNA primers, polymerase δ
fills in the gap,and DNA ligase (not shown) seals the nicks, just as in E. coli

44. FIGURE 10.33 The assembly of new nucleosomes during chromosome replication
requires proteins that transport histones from the cytoplasm to the nucleus and
that concentrate them at the site of nucleosome assembly. PCNA proliferating cell
nuclear antigen

45. ROLLING-CIRCLE REPLICATION
FIGURE 10.30 The rolling-circle mechanism of
DNA replication. Material for progeny
chromosomes (in this case, single stranded
DNA for the virus X174) is produced by
continuous copying around a nicked,
double-stranded DNA circle, with the intact
strand serving as a template. Electron
micrograph courtesy of David Dressler,
Harvard University.

46. ROLLING-CIRCLE REPLICATION
In used
1. By many viruses to duplicate their genome.
2. In bacteria to transfer DNA from donor cells to recipient cell during conjugation.
 The unique aspect of rolling-circle replication is that one parental circular DNA
strand remains intact and rolls (thus the name rolling circle) or spins while serving
as a template for the synthesis of a new complementary strand.
This form of replication is initiated by a break or sequence-specific endonuclease
cleaves in one strand at the origin that creates 3’-OH group and 5’-phosphate
group (termini). New nucleotides are added to the 3’ end used as template.
 As new nucleotides are added to the 3’ end, the 5’ end of the broken strand is
displaced from the template, rolling out like thread being pulled off a spool. The
3’ end grows around the circle, giving rise to the name rolling-circle model.
 The replication fork may continue around the circle a number of times,
producing several linked around the circle, the growing 3’ end displaces the
nucleotide strand synthesized in the preceding revolution.
Eventually, the linear DNA molecule is cleaved from the circle, resulting in a double
-stranded circular DNA molecule & single stranded linear DNA molecule. The linear
molecule circularizes either before or after serving as a template for the synthesis
of a complementary strand.