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DNA REPLICATION

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 DNA Replication
1. DNA replication models
2. Process of DNA replication
A. Lagging and leading strands
B. Synthesis of lagging strand
C. DNA replication in prokaryotes
3. DNA polymerases in prokaryotes and eukaryotes
4. DNA replication in eukaryotes
5. Modes of DNA replication
A. Theta
B. Rolling circle
C. D-loop
D. linear
6. DNA replication at chromosomal tips (telomeres)

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 Importance of DNA replication

What is DNA replication and why is it important?

DNA replication is the synthesis of DNA.

In addition to its central role in ensuring the faithful transmission of genetic

information from one generation to the next, there are additional important reasons for
understanding the process.
•Mutations are generated during DNA replication.
•DNA replication and cell division allow an organism to grow.

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What is DNA replication and why is it important (contd.)?

•DNA replication allows tissue to regenerate.

In some tissues regeneration does not occur because the DNA does not replicate.
Nervous tissue is an example of this. By understanding and manipulating the
process we may eventually be able to control the process.

•Replication is important in the development of diseases such as cancer.

Tumor formation is essentially uncontrolled DNA replication and cell division.

•The DNA replication process represents a potential target for inactivation of

viruses that are not susceptible to metabolic inhibitors such as antibiotics.
The use of AZT in the treatment of HIV infection is a prime example of this.
Inhibiting DNA replication has also been used to control plant virus diseases.

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 DNA replication models
DNA replication is the process by which DNA molecules are copied.

Genetic information must be accurately copied every time a cell

divides.
A single celled human zygote contains 6.4 billion base pairs.
The DNA from the single zygote cell divides to generate
human body made of ~37 trillion cells.
Imagine a consequence if there is an error as low as 1 per
million bases.

How do cells copy DNA?

Proposed DNA replication models:

Conservative replication model
Dispersive replication model
Semiconservative replication mode
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Proposed DNA replication models 1. Semiconservative mode
2. Conservative mode
3. Dispersive mode
1. Semiconservative mode

DNA replication
+

Parental DNA Daughter DNA

The dark blue color represents the original (parental) DNA strand
while the light blue is for the newly made (daughter) strand

After replication both daughter duplexes contain one parental and

one new strand.

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2. Conservative mode

DNA replication
+

Parental DNA Daughter DNA

After replication one daughter duplex is the same as parental DNA but the
another daughter duplex has both strands new.

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3. Dispersive mode

DNA replication
+

Parental DNA Daughter DNA

After replication both daughter duplexes contain segments of parental and

new strands.

Meselson-Stahl experiment (1958) determined the correct mode of DNA replication.

Their strategy involved a technique that could differentiate between parent (old) and
daughter (new) DNA
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A technique to distinguish between heavy and lighter DNA

Nitrogen atoms in DNA come from the

source of nitrogen in the growth medium.
DNA can be labelled with 14N or 15N by
providing appropriately marked nitrogen
source of the growth medium.

In equilibrium density gradient

centrifugation, DNA with different
densities migrates at different rates thus
showing different bands.

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DNA from bacteria After one round of After a second round of Samples taken after
that had been replication, the DNA replication, DNA additional rounds of
grown on medium appeared as a appeared as two bands, replication appeared as
containing 15N single band at one light and the other two bands, as in part c.
appeared as a intermediate weight. intermediate in weight.
single band.

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DNA synthesis
The enzyme DNA polymerase makes DNA.

1. DNA polymerase can’t synthesize DNA without an existing free 3`OH end which
is supplied by a primer; DNA synthesis is initiated with a primer.

2. Primer, a short oligonucleotide, is made of ribonucleotides not

deoxyribonucleotides

3. DNA polymerase uses dNTPs as substrates; NOT NTPs.

Primer dATP
+ dCTP DNA Newly synthesized
DNA + dGTP polymerse strands
(parent) dTTP

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The process of DNA replication (synthesis)
Requirements of replication
 A template strand
 Raw material: primer, nucleotides (dATP, dCTP, dGTP, dTTP)
 Enzymes and other proteins

5` 3`
3` 5` 1. The two strands of DNA must first
Separation of two strands + separate so that a single strand can
Primer binding be used as a template.
5` 3`
3` 5`
2. An RNA primer provides the
5` 3` 3`end for DNA polymerase to add
3` 5`
bases complementary to the
DNA synthesis template strand.
5` 3`
3` 5` 3. DNA synthesis takes place only in
5`-3` direction
5` 3`
3` 5`

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DNA polymerases in E. coli
Catalytic activities
Type of
5`-3` 3`-5` 5`-3`
DNA Function
Polymerase Exonuclease Exonuclease
polymerase
activity activity activity
I Yes Yes Yes Removes and replaces
primers
II Yes Yes No DNA repair; restarts
replication DNA halts
synthesis
III Yes Yes No Elongates DNA

IV Yes No No DNA repair

V Yes No No DNA repair; translesion

DNA synthesis

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 The process of DNA replication (synthesis)
1. A template strand is required to synthesize a desired sequence of DNA.
2. DNA bases, complementary to the template strand, are added one by one at the 3` end
of DNA; dNTPs act as a source of new nucleotides.
3. The 5`phosphate group of the incoming dNTP attacks the 3`-OH of the last nucleotide
on the strand.
4. A phosphodiester bond links the two nucleotides; two phosphates are released.

Template
5` 3` strand

dNTP

3`
PPi

5`
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Synthesis of two strands of DNA

Origin of replication
5` 3`
3` 5`

At the origin of replication, a part of

DNA separates in two strands to
expose the templates.

RNA primers are synthesizes by the

enzyme primase.

DNA polymerase adds bases at the 3`

end of the growing chain.
The added bases are complementary
to the template strand.

In the open duplex a part of DNA is shown unreplicated. Why? (16)

 The replication fork moves further on
both ends.

It allows synthesis of new RNA

primer on the unreplicated part of the
template. DNA polymerase now can
add bases at the 3` end of the RNA
chain.

As the replication fork opens further

in both directions, two strands
d (marked region‘c’) get continuously
c synthesized while the other two
(marked region‘d’) remain
unreplicated and wait for a new
d c primer. (17)
 c d As the replication fork opens further in
both directions, new primers are
synthesized in two strands (marked
c region ‘d’) and DNA gets synthesized.
d
Leading strand
DNA synthesis from region ‘c’
5` 3` template is continuous; it does not
3` 5` require new primer at further opening
of replication fork.
This template strand is called the
Leading strand
(continuous synthesis) leading strand.

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 c d

d c

5` 3`
3` 5`
Lagging strand
(discontinuous Okazaki Leading strand
synthesis) fragments

Lagging strand
• For the template in region ‘d’, the enzyme cannot add bases at the 5` end so
DNA synthesis re-starts at the fork; this happens each time DNA unwinds.
• The discontinuous synthesis, involving the production of small fragments
(called Okazaki fragments), is a slower process than the synthesis of the other
strand.
• This strand is called the lagging strand.
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DNA synthesis in bacteria
The whole process of DNA synthesis will be discussed through following key points

1. Initiation

2. Unwinding
i. DNA helicase
ii. Single strand binding proteins
iii. DNA gyrase

3. Elongation
i. Synthesis of primers
ii. DNA synthesis by DNA polymerase III
iii. DNA synthesis by DNA polymerase I
iv. DNA ligase

4. Termination

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DNA synthesis in bacteria
1. Initiation: Initiator proteins
(DnaA)
• DNA replication starts at a
specific point called origin of
replication.

• Bacterial chromosomes are

replicated through a single origin
of replication.

• In E. coli the oriC region is made

of 245 bp where the initiator
protein DnaA binds.
ssDNA binding
proteins
• By binding of DnaA at oriC, a
short region of DNA unwinds that
allows helicase and ssDNA Helicase
binding proteins to bind.

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DNA synthesis in bacteria
2. Unwinding
The DNA synthesizing enzymes require single stranded DNA template. It is achieved with
the help of a number of proteins that unwind and stabilize DNA in single stranded form.
These proteins include,
DNA helicase (DnaB): it breaks the hydrogen bonds between two strands
Single-strand-binding proteins: they keep single stranded DNA stable
DNA gyrase: a topoisomerase II that controls the supercoiling, it reduces the torque that
builds up ahead of the replication fork.
Nalidixic acid, an antibiotics that is used for controlling bacterial infections, works by
binding to DNA gyrase and stopping DNA replication.

Origin of
DNA gyrase replication Single-strand-binding proteins

unwinding DNA helicase unwinding

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DNA synthesis in bacteria (continued)
3. Elongation
i. Synthesis of primers
DNA polymerases cannot initiate DNA synthesis on a bare template, it requires a free
3`-OH group to add a new nucleotide.
A 10-12 nucleotide long RNA primer, synthesized by an enzyme called Primase
(DnaG), provides a free 3`-OH group to the DNA polymerase.

ii. DNA synthesis by DNA polymerase III

Once DNA is in a single-stranded form and a primer has been added, DNA polymerase
starts synthesizing DNA.
DNA polymerase III is the main enzyme doing bulk of DNA replication. It has two
catalytic activities-
 5`-3` polymerase activity- adds nucleotides to the 3` end of the growing chain.
 3`-5` exonuclease activity-removes wrong bases.
Other DNA polymerases present in bacteria are listed in a table.

iii.Synthesis of DNA by DNA polymerase I

DNA polymerase I with its 5`-3` exonuclease activity removes the ribonucleotides
(RNA primer) and synthesizes DNA.

iv. DNA ligase

The breaks in the DNA chain are sealed by the enzyme DNA ligase.
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 RNA DNA Primase
DNA ligase Primer
DNA polymerase

Topoisomerase
DNA polymerase
Helicase
By DNA_replication_en.svg: LadyofHats Mariana Ruizderivative ssDNA binding
work: Vojtech.dostal (talk) - DNA_replication_en.svg, Public Domain,
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proteins

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 DNA synthesis in bacteria (continued)
4. Termination

There are different ways of termination in different types of DNAs

Replication terminates when two replication forks meet
Some DNA use specific termination sequences called Ter sites. A Tus protein binds at the
ter site and causes termination of replication.

Summary of proteins involved in DNA replication

1. Initiator protein: DnaA binds at oriC to initiate replication
2. DNA helicase: DnaB unwinds DNA at replication fork.
3. Single-strand-binding (SSB) proteins: binds to ssDNA to prevent reannealing.
4. DNA gyrase: moves ahead of the replication fork, relieves torque.
5. DNA Primase: Synthesizes a short RNA primer to provide a 3'-OH group for the
attachment of DNA nucleotides.
6. DNA polymerase III: synthesizes DNA by extending the RNA primer. It leaves the
DNA as it touches the 5`end of the previously made fragment.
7. DNA Pol I: removes RNA primers and replaces them with DNA.
8. DNA ligase: joins Okazaki fragments by sealing breaks in the sugar–phosphate
backbone of newly synthesized DNA.
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Modes of replication
• DNA is copied by semiconservative replication in all organisms.

• Replicon is an individual unit of DNA with an origin of replication.

• Origin of replication is the point where replication starts.

• Eukaryotic chromosomes have multiple origins of replication, in contrast to

bacteria that have a single origin to replicate the whole chromosome.

Modes of DNA replication

1. Theta replication
2. Rolling circle replication
3. D-loop replication
4. Linear replication
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1. Theta replication
John Cairns in 1963 provided a visible 2. Cells were grown in (3H) thymidine for
evidence of theta replication by growing another round of replication for a short
cells in radioactive growth media. time intervals and autoradiographed

1. DNA from cells grown in (3H)

thymidine for one replication cycle
were autoradiographed.
Both strands
This autoradiograph are radioactive
showed that the
bacterial chromosome
is circular

In the semiconservative mode of

replication, one whole strand will be These moon shaped patterns (called theta
radioactive (light blue) and the other structures) of various sizes were seen
strand will remain non-radioactive.
This is known as theta mode of DNA
replication.

This experiment also proves that DNA

replicates in a semiconservative mode
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2. Rolling circle replication

Some plasmids, bacteriophages and viral DNA replicate

through unidirectional replication.
A single circular DNA produces multiple copies of the genome.
A
Replication starts with a nick at the origin of replication (B).
DNA polymerase binds to the nick and starts adding nucleotides
A nick at
the origin at the 3` end, the old strand is displaced (C)

B Polygenomic tail

Old DNA The polygenomic tail (D) can be cut to give

new DNA
synthesized displaced unit genome length or it can be replicated to
the double stranded form and then cleaved

C D

Rolling
direction of
the circle (28)
3. D-loop replication

Replication of mitochondrial and chloroplast DNA initiates with the formation of a

D-loop (displacement loop)
1. One strand (The L strand) is displaced to allow synthesis of the primer on the H
strand. Primer synthesis on the L strand does not take place at this stage.
2. The primer is extended to replicate the H strand.
3. When the H strand is nearly complete then replication of the L strand initiates
with the synthesis of a primer.

Primer
D-loop
H-strand
L-strand

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4. Linear replication
DNA replication in Eukaryotes

Specific challenges in eukaryotic DNA replication

1. DNA template is associated with histones vs no packaging proteins in
prokaryotes
2. Genomes are larger vs much smaller size in prokaryotes
3. Chromosomes are linear vs circular in prokaryotes

1. DNA template is associated with histones vs no packaging

proteins in prokaryotes
Eukaryotic DNA is packed with histones, so replication will require disruption of the
chromatin structure and the reassembly of nucleosomes to pack newly synthesized DNA.
The disruption of the chromatin structure occurs by the replication fork.
The nucleosomes on each daughter DNA molecule have a mixture of histones from the old
nucleosomes and newly synthesized proteins.

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2. Genomes are larger vs much smaller size in prokaryotes
Eukaryotes have multiple origins of replication. It makes the process of DNA
replication faster for the large eukaryotic chromosomes.

Replication bubble
~10,000-100,000 replication origins in a human somatic cell.

A number of DNA polymerases (see Table 12.5 on the next slide) are involved in
DNA synthesis.
The three polymerases alpha, delta and epsilon carry out nuclear DNA replication

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 TABLE 12.5 DNA polymerases in eukaryotic cells
5' 3' 3'  5'
DNA Polymerase Exonuclease
Polymerase Activity Activity Cellular Function
α (alpha) Yes No Initiation of nuclear DNA synthesis and DNA repair; has
primase activity
δ (delta) Yes Yes Lagging-strand synthesis of nuclear DNA, DNA repair,
and translesion DNA synthesis
ε (epsilon) Yes Yes Leading-strand synthesis
γ (gamma) Yes Yes Replication and repair of mitochondrial DNA
ϕ (zeta) Yes No Translesion DNA synthesis
η (eta) Yes No Translesion DNA synthesis
υ (theta) Yes No DNA repair
ι (iota) Yes No Translesion DNA synthesis
κ (kappa) Yes No Translesion DNA synthesis
λ (lambda) Yes No DNA repair
µ (mu) Yes No DNA repair
σ (sigma) Yes No Nuclear DNA replication (possibly), DNA repair, and
sister-chromatid cohesion
φ (phi) Yes No Translesion DNA synthesis
Rev1 Yes No DNA repair
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3. Eukaryotic chromosomes are linear vs circular in prokaryotes
There is an inherent problem in replicating
the ends of linear chromosome Old DNA
New DNA
RNA primer
DNA replication of the top strand is 5` 3`
shown in B, C and D.
3`
(A)
5`
First the RNA primers are
degraded (C).
 DNA polymerase fills gaps
by extending DNA (B)
synthesis. All internal gaps RNA primer gets degraded
are filled (D).
leaving gaps

The terminal gap can not be filled by (C)

DNA polymerase (why?), leaving
tips single stranded. All internal gaps are filled
but not the terminal gap
It will cause shortening of the
chromosome. (D)
Bases at the 3` end of the
template are not replicated
Circular chromosomes don’t face this situation (why?) (33)
3A. The enzyme telomerase replicates chromosome tips

 The tips of chromosomes have specialized DNA called telomeres.

 Telomeres are made of 100 to1000 repeats of a small sequence.

 In humans and other mammals, the telomeres have a sequence TTAGGG which
is repeated several times.
 The repeats do not code for an RNA or a protein product but have an important
role in DNA replication.

 The repeats are added by an enzyme called telomerase, a reverse transcriptase.

 The telomerase carries a small RNA molecule which acts as a template to
synthesize the ‘repeat’

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The enzyme telomerase is a reverse transcriptase

In tetrahymena the telomeres have repeats of 5`-TTGGGG-3` and

the telomerase contains RNA with a sequence 3`-AACCCC-5`.

The ‘repeats’ are synthesized by telomerase in two steps:

(a) extension of 3` overhangs and
(b) synthesis of the complementary strand.

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Telomere lengthening
RNA component 3` AACCC
5` TTGGGGTTGGGGTTGGGG
AACCCCAACCCC Telomerase binds to
the 3` overhang
Telomerase AACCC
3` AACCCCAACCCC
(a) Extension of the 3` overhang 5` TTGGGGTTGGGGTTGGGG

Telomerase containing an RNA Elongation

component binds to the 3`
overhang.
3` AACCC AACCCCAACCCC
5` TTGGGGTTGGGGTTGGGGTTGGGG
Telomerase uses RNA as a template
and synthesizes DNA.
(This enzyme activity is known as Translocation
RNA dependent DNA polymerase or
reverse transcriptase) 3` AACCC AACCCCAACCCC
5` TTGGGGTTGGGGTTGGGGTTGGGG
Once the RNA template is
replicated, telomerase translocates
making the template available for
Elongation
further DNA synthesis.
3` AACCC AACCCCAACCCC
5` TTGGGGTTGGGGTTGGGGTTGGGGTTGGGG
This way telomerase can extend the
3` end without the template strand. (36)
(b) Synthesis of the complementary strand
3` AACCC AACCCCAACCCC
5` TTGGGGTTGGGGTTGGGGTTGGGGTTGGGG
The 5` end of the tip is synthesized Telomerase leaves
by conventional replication. DNA
As the telomerase leaves, the DNA 3` AACCC
polymerase α synthesizes an RNA 5` TTGGGGTTGGGGTTGGGGTTGGGGTTGGGG
primer and then DNA is synthesized. Primase synthesizes an RNA
primer
The RNA primer is later removed 3` AACCC ACCCC
leaving a small overhang at the 5` 5` TTGGGGTTGGGGTTGGGGTTGGGGTTGGGG
end.
DNA polymerase fills the gap
by synthesizing DNA
3` AACCCCAACCCCAACCCCAACCCCAACCCC
5` TTGGGGTTGGGGTTGGGGTTGGGGTTGGGG
The RNA primer gets
degraded with time
3` AACCCCAACCCCAACCCCAACCCCA
5` TTGGGGTTGGGGTTGGGGTTGGGGTTGGGG

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3B. Some DNA ends are replicated by using protein primers

5` 3`
3` 5`
5`
OH OH Protein with free OH group

5`
5` 3`
3` 5`
New DNA
5` 3`
3` 5`

This is an example where a non-nucleic acid molecule primes DNA synthesis to replicate
linear DNA.

Protein primers are used by several viruses ( including bacteriophage Phi 29 and mammalian
viruses adenovirus and poliovirus) linear plasmids and chromosomes of Streptomyces.

A protein binds at the 3` end displacing the complementary strand.

The protein has a 3` OH free for priming DNA synthesis. DNA polymerase synthesizes DNA.
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1. In a Meselson-Stahl type experiment, bacterial cells grown in 14N medium for
many generations were washed and then allowed to grow in 15N medium.
DNA was extracted just after transfer (0 generation) and then after 1 and 2
generations. All these samples were analyzed by CsCl gradient
centrifugation. From DNA isolated from generations 0, 1 and 2 show
A. Position of bands in the centrifuge tubes (10 marks) and
B. 14N and 15N labelled DNA strands (10 marks)

Answer both questions for each mode of replication ie semiconservative,

conservative and dispersive mode of replication

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1. Bacterial cells grown in 15N medium for many generations were
washed and then allowed to grow in 14N medium. DNA was
extracted just after transfer (0 generation) and then after 1 and 2
generations. All these samples were analyzed by CsCl gradient
centrifugation. Which of the following statements is NOT true?
a. Generation 0 sample will have only one band
b. Generation 1 sample will have only one band
c. Generation 2 sample will have two bands
d. At least one band in generation 0 and 1 samples will be of the
same density
e. At least one band in generation 1 and 2 samples will be of the
same density

2. Which of the following is not a substrate for DNA polymerase

a. dATP
b. dCTP
c. dTTP
d. dUTP
e. dGTP

3. The major product of DNA replication is

a. DNA
b. RNA
c. Protein
d. DNA and RNA
e. DNA and protein
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On the following drawing identify
I. Origin of replication (oriC)
II. The polarity of all template strands and newly synthesized strands
III. Leading and lagging strands
IV. Okazaki fragments
V. Locations of primers

Can you put the following in an order as they happen during DNA replication?
1. Sealing of nicks by DNA ligase
2. Control of supercoiling by DNA gyrase
3. Formation of Okazaki fragments
4. Separation of two strands by DNA helicase (DnaB)
5. Unwinding of two strands at oriC
6. Synthesis of primers by primase
7. DNA synthesis by PolIII
8. DNA synthesis by PolI
9. Binding of ssb proteins
10. DnaA binding at oriC
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1. Why is an RNA primer get synthesised in DNA replication?

2. DNA polymerase cannot initiate DNA synthesis but it can elongate an existing chain.
How is the initial nucleotide chain get synthesized?

3. Differentiate between leading strand and lagging strand.

4. What are Okazaki fragments? Why are they formed?

5. DNA Pol I and Pol III both can synthesize DNA, will there be any problem if Pol I is
unavailable?

(42)
What is the role of the following in DNA replication?
oriC

DNA helicase

ssb

DNA gyrase

Primase

5’ to 3’ exonuclease activity

5’ to 3’ polymerase activity

3’ to 5’ exonuclease activity

(43)
In John Cairn’s experiment E. coli cells grown in (3H) thymidine for one replication cycle
were autoradiographed, the image is shown below

What did they conclude from this result?

When these cells were grown in (3H) thymidine for another round of replication for a
short time interval and autoradiographed, the image looked like as below

What did they conclude from this result?

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List the four modes of DNA replication. Provide an example for each mode of
DNA replication.

What is the basis behind naming the following modes of replication

Theta
Rolling circle
D-loop
Linear

Give an example of DNA that is replicated by D-loop mode.

Where do you find the replication of linear DNA molecules?

(45)
In comparison to prokaryotes, what are specific challenges in
eukaryotic DNA replication?

What strategy do eukaryotes use to replicate their large genome in

timely manner?

What happens to nucleosomes during DNA replication?

(46)
There is an inherent problem in replicating the ends of linear chromosomes. The RNA
primers that initiate DNA replication are degraded and the gaps are filled by a DNA
polymerase that fills gaps by extending DNA synthesis. All internal gaps are filled but the
terminal gap can not be filled by DNA polymerase, leaving tips single stranded. If not
fixed, it will cause shortening of the chromosome. The following figure shows the newly
synthesized strands have several bases missing at one end.
a b
c d
DNA replication
a b
e f
g h
c d
To avoid loss of terminal sequences different strategies are used by E. coli, linear prokaryotic genomes,
and eukaryotic genomes.

A) What strategy does E. coli use?

B) What strategy do linear prokaryotic genomes use?
C) What strategy do eukaryotic genomes use?

The 5` and 3 ends of the parent and daughter DNA strands are shown by letters a-h.
Name all letters that represent the 5` end-
Name all letters that represent the 3` end-
Which end has the missing bases in the newly synthesized strands? (47)
1. In a linear fragment of DNA the ends can not be replicated by DNA polymerases.
Eukaryotes use telomerase enzyme to replicate chromosome tips. This enzyme has
a. DNA polymerase activity
b. RNA polymerase activity
c. Reverse transcriptase activity
d. DNA ligase activity
e. DNA helicase activity

1. In tetrahymena the telomeres have repeats of 5`-TTGGGG-3`. What will be sequence of

RNA in the telomerase?

2. If DNA polymerase is a DNA dependent DNA polymerase what type of enzyme is

Telomerase?

3. DNA polymerases can add nucleotides only to an existing 3`-OH group. Provide
different examples that provide the free 3`-OH group for DNA polymerases.

4. Linear chromosomes have been seen in prokaryotes as well. How do they replicate
DNA tips? Do they have telomerases?

(48)