Block 2 DNA Replication

UNIT 5
INTRODUCTION TO
REPLICATION

Structure
5.1 Introduction Unidirectional and Bidirectional
replication
Expected Learning Outcomes
5.5 Semidiscontinous Model of
5.2 Chemistry of DNA
DNA Replication
Polymerization
Okazaki Fragments
Rationale of Priming
Replication Fork
Phosphodiester Bond Formation
Leading and Lagging Strands
5.3 Modes of DNA Replication
5.6 Fidelity of DNA Replication
DNA Replication is
Semiconservative: Experimental 3´→5´ Exonuclease Activity
Proofs
5.7 Summary
Semiconservative Replication in
5.8 Terminal Questions
Eukaryotes
5.9 Answers
5.4 Origin of Replication

5.1 INTRODUCTION
In the previous block you studied about DNA organization beginning from the
architecture of chromatin to how it is packaged into highly compact
chromosomes. As a carrier of genetic information, DNA performs two types of
functions: autocatalytic and heterocatalytic. This unit deals with DNA
replication, an autocatalytic function of DNA, by which it gives rise to multiple
copies of itself. Heteroctalytic functions are the ones by which it directs the
94 synthesis of RNA and proteins.
Unit 5 Introduction to Replication
In this unit you shall learn about the types and principles of replication. Before
commencing this unit, we advise you to revise Unit 13 Nucleic acids (Block IV,
BBCCT-101). It would help you to understand this processes better when you
have a clear picture in your mind about the structural aspects of DNAs.

Expected Learning Outcomes

After studying this unit, you should be able to:

 explain the formation of phosphodiester bond;

 differentiate between conservative and semiconservative replication;

 discuss dispersive replication;

 explain the significance of Okazaki fragments; and

 comment on the importance of proof reading.

5.2 CHEMISTRY OF DNA POLYMERIZATON

Every living cell has a genetic program that controls the synthesis of all other
biomolecules in the cell. DNA is the genetic material in most of the organisms
while RNA acts as genetic material in some viruses. The genetic material has
to satisfy four important characteristics i.e. it must be able to synthesize exact
replica of itself in a process called as DNA replication; it must express the
stored information in a process called as DNA transcription, it must be able to
store information and stored information must undergo stable heritable
changes called mutations.

In this perspective it becomes imperative to recall the central dogma of

molecular biology. There are three different processes viz. replication,
transcription and translation responsible for the flow of genetic information
popularly known as the central dogma of molecular biology Fig. 5.1.
Replication is one amongst that and is important for the inheritance of genetic
information and for its conversion from one form to another. During
transcription, message stored in DNA is transcribed and transferred into single
stranded sequence of RNA and later translated into proteins. You will study
about these processes in detail other courses. You already know that DNA is a
self replicating molecule; however, H.M. Temin and D. Baltimore in 1970
modified this information flow and highlighted the process of reverse
transcription about which you will study later. In retroviruses and
retrotransponsons, reverse transcriptase, an enzyme also known as RNA-
directed DNA polymerase catalyses the transcription of retrovirus RNA into
DNA.

Fig. 5.1: Central dogma of molecular biology highlighting key processes to

sustain life. 95
Block 2 DNA Replication

As you know, synthesis of a daughter duplex DNA molecule identical to the

parental duplex DNA is called as DNA replication. The method of replicating
DNA can be easily understood from its structure. Two strands of double helical
DNA are complementary. Watson and Crick first suggested that one strand
act as template for the synthesis of another. Thus DNA replication produces
two “daughter” duplexes each of which contains one “parental” and one “new”
strand as shown in Fig. 5.2.

Fig. 5.2: Possible method of DNA replication as suggested by Watson and Crick.

In 1957 Arthur Kornberg reported first enzyme which can synthesize DNA in
E. coli called as DNA polymerase I (DNAP I). DNAP I is a single polypeptide
enzyme, requires template, primer, four dNTPs and Mg++. Later on more such
DNA polymerizing enzymes were reported and these were called as DNAP II,
DNAP III, DNAP IV, DNAP V and so on. The following reaction of DNA
synthesis is catalyzed by DNAP I.

(dNMP)n + dNTP (dNMP)n+1 + PPi ΔG°‘ = 2kJ/mol

Lengthened DNA
PPi 2Pi…………………………… ΔG°' = -30kJ/mol

Net ΔG°' = -28kJ/mol

5.2.1 Rationale of Priming

DNA polymerases cannot polymerize DNA de novo. All DNA polymerases
require a primer i.e. a short stretch of single stranded deoxyribo or
ribonucleotides about 20 nucleotides in length having a free 3´ OH group in
order to carry out DNA synthesis. Further chain elongation takes place at the
96 3´ end of this primer in 5´-3´ direction in an antiparallel manner.
Unit 5 Introduction to Replication
5.2.2 Phosphodiester Bond Formation
As you know that the template strand is copied from 3´ to 5´ direction while
DNA synthesis takes place from 5´ to 3´. The new nucleotide is selected from
a pool of nucleotides by Watson Crick base pairing rule between incoming
nucleotide and template nucleotide. Means if there is A at template nucleotide,
then T will be selected as incoming nucleotide and vice versa or if G is the
template nucleotide, then C will be selected as incoming complementary
nucleotide and vice versa.

During polymerization reaction, free 3´ hydroxyl group of the last nucleotide at

the 3´ end of the growing chain attacks the alpha phosphorus of the incoming
nucleotide thus making a phosphodiester bond and PPi is released (Fig. 5.3).
This reaction is endergonic and thermodynamically unfavourable.

Fig. 5.3: Chemistry of DNA synthesis.

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Block 2 DNA Replication

This PPi will be hydrolysed by phosphohydrolase (phosphatase) thus forming

Pi. The energy is released in this process and overall the reaction of DNA
synthesis is made thermodynamically feasible. Thus, the energy required for
the addition of new nucleotide is contributed by the incoming monomeric
nucleotide. Such reactions are called tail growth reactions and they can
undergo proof reading thereby meaning that if last added nucleotide is wrong,
then it can be replaced by a correct nucleotide.

5.3 MODES OF DNA REPLICATION

Three different models have been suggested for replication of DNA:
conservative, semi-conservative and dispersive as shown in Fig. 5.4.

During DNA replication, double helix acts as a template for the synthesis of
new double helix. Conservative model suggests that after one round of DNA
replication, old duplex is conserved and new duplex is composed of two
completely new strands.

As per semi-conservative model, each of the two daughter duplexes

consists of one old and one new strand (Look at Fig. 5.4). The DNA double
helix is unwound at the origin of replication and replication occurs in the
antiparallel direction. This is the mechanism of DNA replication in all cells.

According to dispersive model, sections of the old duplex are dispersed

somewhat randomly to the two daughter duplexes. During dispersive
replication, the original DNA strand breaks and new DNA strand recombines in
a random fashion involving parts of parental strand and newly synthesized
strand.

98 Fig. 5.4: Three possible modes of DNA replication.

Unit 5 Introduction to Replication
5.3.1 DNA Replication is Semi-conservative:
Experimental Proofs
DNA replication occurs by semi-conservative model. This was proved by
Matthew Meselson and Franklin Stahl in 1958. They used E. coli as their
research organism and these cells were incubated with naturally occurring
Nitrogen (N14) and heavy but non radioactive isotopes of nitrogen (N15) to
label DNA with different densities as nitrogen is a component of bases. When
E.coli cells are incubated with 15NH4Cl for several cell generations, DNA will be
N15 labeled and thus will be having heavy density. When such E. coli cells
were harvested, lysed and DNA separated on CsCl Density Gradient
Centrifugation, then a single band of heavy density was observed, confirming
that DNA was N15 labeled. During CsCl Density Gradient Centrifugation,
solution of CsCl is centrifuged at very high speed thus creating a gradient of
CsCl having maximum density at the bottom and minimum density at the top
of the tube. When two molecules of different densities are loaded onto such
tubes, then these molecules will rest at a point when their densities match with
the density of the medium, thus separating the molecules as shown in Fig. 5.5.

Fig. 5.5: CsCl Density Gradient Centrifugation.

Such N15 labeled cells were transferred to N14 medium for one cell generation
and E. coli cells were harvested, lysed and DNA separated on CsCl Density
Gradient Centrifugation. After centrifugation a single band of intermediate
density (N15 N14) was observed (Fig. 5.5) thus confirming that DNA replication
occurs by either semi-conservative or dispersive model. These observations
reject the conservative model as in this model two bands (one light density
and other heavy density) would have been observed.

Such N14 labeled cells were then transferred to N14 medium for one more cell
generations and E. coli cells were harvested, lysed and DNA separated on
CsCl Density Gradient Centrifugation. Here one band of low density (N14 N14)
and another band of hybrid density (N15 N14) was observed, thus confirming
that DNA replication occurs by semi-conservative model (Fig. 5.5). These
observations further rejected the dispersive model as both the bands of
intermediate densities would have been observed if the replication would have
occurred by dispersive mode as shown in Fig. 5.6. 99
Block 2 DNA Replication

Fig. 5.6: Semi-conservative mode of DNA replication.

These experimental evidences clearly prove that DNA replication occurs

bysemi conservative mode.

5.3.2 Semi-conservative Replication in Eukaryotes

In eukaryotes replication occurs in semi-conservative mode. It was proved by

J. Herbert Taylor, Philip Woods and Walter Hughes in 1957 in Bean plant
(Vicia faba). They exposed root tips of the plant to Thymidine (H3) and this
radioactive precursor labeled both the sister chromatids in a chromosome.
Then autoradiography was performed. During anaphase, labeled chromatids
go to opposite poles. Second round of replication in the absence of labeled
thymidine gives only one labeled chromatid in each chromosome. This also
provided evidence for semi-conservative replication. Now guess what would
have been their results if replication had been conservative or dispersive.
100 Effects of recombination can also be observed as shown in Fig. 5.7.
Unit 5 Introduction to Replication

Fig. 5.7: Semi-conservative mode of DNA replication in eukaryotes.

SAQ 1
a) State whether the following statement is true of false:
i) DNA replication occurs by conservative model in prokaryotes.
(True/False)
15
ii) N is radioisotope of Nitrogen. (True/False)
iii) Nitrogen is the component of DNA in the form of nitrogenous
bases. (True/False)
iv) One strand of DNA acts as a template for the synthesis of
complementary strand during DNA replication. (True/False)
b) Fill in the blanks:
i) Synthesis of daughter duplex DNA molecules identical to the
parental DNA molecule is called………
ii) Synthesis of DNA using RNA as a template is called………..
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 Block 2 DNA Replication

5.4 ORIGIN OF REPLICATION

Replication of DNA is important to all the organisms. It does not begin at the
Theta structure
terminal position; rather it always begins at an intercalary position. Replication
One complete dotted
starts at a point on the chromosome where the two parental strands begin to
circle is formed by
radioactivity separate. This point of separation/unwinding is called “origin of replication”.
incorporated during They are called as Ori sequences in prokaryotes. In bacteria, DNA is small so
one round of DNA there is single Ori sequence. But in eukaryotes, multiple origins are there as
replication (the chromosomes are big.
second stand is not
visible as it is not Region of the DNA replicated from a single origin is called a replicon. Thus
labeled). Two dotted
there is one replicon per chromosome in prokaryotes. E. coli replicon is 4.2
partially completed
circles on the top million bp. At each origin, two replication forks (region of the DNA where
indicate the replicated DNA meets the unreplicated DNA) are formed. These replication
replication completed forks move in opposite directions and thus DNA replication is generally
in the second round
bidirectional (Fig. 5.8). Termination of replication takes place at specific
of DNA replication
(there is single sequences called ter sequences.
dotted circle on the
top as its template
stand is non
radioactively labelled
so it is not visible).

Fig. 5.8: Origin of DNA replication, its termination and replication fork.

Here you must note that once the origin of replication is identified and
replication fork is formed, then DNA replication may proceed from this point
unidirectionally or bidirectionally.

5.4.1 Unidirectional and Bidirectional Replication

In unidirectional replication, growth proceeds along both the strands in the
same direction leading from the origin (Fig. 5.9). Since the replication fork
moves in one direction from the origin, this type of replication is called
102
Unit 5 Introduction to Replication
unidirectional. Some types of bacteria use this type of mechanism. In
bidirectional replication two replication forks are formed at the origin and these
move away from the origin in both the directions as parental double helix is
separated (Fig. 5.9). This mechanism is employed in all prokaryotes and
eukaryotes.

Fig. 5.9: Diagrammatic representation of unidirectional and bidirectional

replication.

5.5 SEMI DISCONTINOUS MODEL OF

DNA REPLICATION
Semi-discontinuous nature of DNA replication means that one strand is
synthesized continuously whereas the other strand is synthesized
discontinuously.

5.5.1 Okazaki Fragments

As mentioned above, Okazaki fragments are short, newly synthesized DNA

fragments that are formed on the lagging template strand during DNA
replication. Okazaki fragments are 1000 to 2000 nucleotides long in E. coli and
are 100 to 200 nucleotides long in eukaryotes. They are separated by RNA
primers and are unligated until RNA primers are removed. Enzyme ligase
connects the two Okazaki fragments into one continuous newly synthesized
complementary strand.

5.5.2 Replication Fork

Replication fork is a structure that forms during DNA replication within the
nucleus. Its formation is initiated by helicases, an enzyme which separates
the two DNA strands by breaking the hydrogen bonds holding them together.
The resulting structure has two branching "prongs", each one made up of a
single strand of DNA that acts as a template. Single strand binding proteins
bind to DNA strands and prevent the DNA double helix from re-annealing after
DNA helicase unwinds it, thus maintaining the strand separation. Both the
strands serve as template and are referred as leading and lagging strands.
103
Block 2 DNA Replication

Fig. 5.10: Structure of replication fork.

5.5.3 Leading and Lagging Strands

The strand synthesized by 5´-3´ polymerization in the direction of movement of
replication fork is called leading strand and the strand synthesized by 5´-3´
polymerization in the direction opposite to the movement of replication fork is
called lagging strand. DNA synthesis is continuous on leading strand and
it is discontinuous on lagging strand. The discontinuous synthesis of DNA
on lagging strand was discovered by Reiji Okazaki, a Japanese scientist
(Fig. 5.11 ).

104 Fig. 5.11: Synthesis of the leading and lagging strands.

Unit 5 Introduction to Replication
Pulse-chase experiments were conducted to prove discontinuous DNA
synthesis. E. coli cells were allowed to grow in the presence of 3H
deoxythymidine and then cells were harvested and lysed. DNA was isolated
and denatured. Then it was separated on the basis of size. It was observed
that half of the radioactivity was present in very long DNA molecules because
of continuous DNA synthesis. Rest half of the activity was present in small
DNA pieces of about 1000-2000 nucleotides long because of discontinuous
DNA synthesis on lagging strand.

Later on these pieces were joined together by DNA polymerase I (by utilizing
its 5´-3´ polymerization and 5´-3´ exonuclease activity) and DNA ligase I and
thus very large DNA molecules are formed with time Fig. 5.12.

Fig. 5.12: Discontinuous DNA synthesis on lagging strand.

SAQ 2
a) Define the following:
i) Origin of replication
ii) Bidirectional replication
iii) Okazaki fragments
iv) Replication fork
b) State whether the following statement is rue of false
i) Replication begins at the terminal position on the chromosome.
(True/False) 105
Block 2 DNA Replication
ii) Termination of replication takes place at specific sequences called
ter sequences. (True/False)
iii) In some bacteria unidirectional mechanism of replication occurs.
(True/False)
iv) Single strand binding proteins bind to RNA and prevent
degradation. (True/False)
v) Pulse-chase experiments used 3H deoxythymidine to prove
discontinuous DNA synthesis. (True/False)

5.6 FIDELITY OF DNA REPLICATION

In the previous section we learnt about semi conservative model of DNA
replication. Now we will learn about fidelity of DNA replication. DNA cannot
afford to have mistakes during DNA replication as it is the master molecule of
the cell. A high degree of accuracy is maintained during DNA replication, as
even a small mistake (1 per 10000 nucleotides) will cause enough mutations
in any gene which may alter the function of a protein or can even make it
completely functionless.

DNA polymerases are very accurate enzyme as the base composition of

product is found similar to that of the template. There are various levels to
maintain accuracy during DNA replication. In E.coli, during DNA replication,
one mistake may get incorporated for every 109 to 1010 added nucleotides. So
for E.coli chromosome of 4.7x106 bps, one error is made for 1000 to 10,000
rounds of replications.

First level of maintaining accuracy during DNA replication is the correct pairing
between complementary nucleotides as per Watson-Crick base pairing rule.
The DNA polymerase adds a nucleotide to the available 3´-OH end of a
growing DNA strand or RNA primer, only if the prior nucleotide is properly
base paired with the template nucletoide. If a mismatched nucleotide is
present, growth of the strand is temporarily halted. The nucleotide base-pair
wrongly inserted in the growing strand is excised by 3´-5´ exonuclease activity.
This is known as a proof reading function of DNA polymerase. With a correct
3´ end established, elongation by DNA polymerase is resumed until the
template is completely replicated. However DNA polymerases insert one
incorrect nucleotide for every 104 to 105 correct ones as these mistakes are
because of unusual tautomeric form of a base. The bases like adenine and
cytosine exist in amino and imino forms, while bases guanine and thymine
exist in keto and enol forms. This allows them to hydrogen bond with incorrect
partner.

Thus, accuracy of polymerization is further improved by 102 to 103 fold by

3´→5´ exonuclease activity of DNA polymerase. Accuracy rate is further
improved and brought to the level of 109 to 1010 by a mismatch repair system
that repairs mismatches incorporated during DNA replication about which you
will study in Unit 12. Let us understand in detail the proofreading activity or
106 5´→3´ exonuclease activity of DNA polymerase I.
Unit 5 Introduction to Replication
DNA polymerase I (DNAP I) adds nucleotides in the 5´ to 3´ direction one at a
time. DNAP I has three activities: 5´→3´ exonuclease activity which removes
RNA primers at the end of each Okazaki fragment, 5´→3´ polymerase activity
which fills the gap between Okazaki fragments, and 3´→5’ exonuclease
activity which functions in proofreading.

5.6.1 3´→5´ Exonuclease Activity

This activity improves the overall accuracy of DNA replication by proofreading
at the 3´ end of the DNA molecule. If any wrong nucleotide is added at the 3´
end of newly replicated DNA molecule, then it will halt the process of
replication and needs to be replaced. For example, there is A in the template
stand, then T should be selected by enzyme as per Watson-Crick base pairing
rule. But C attains a rare tautomeric form called C*, which can pair with A. So
enzyme will select C* opposite to A and it will be incorporated into the growing
DNA strand. But as soon as the enzyme polymerizes C*, it will change back to
normal form C. So now there is a mismatch in the DNA molecule. Thus, DNA
polymerase will use its 3´→5´ exonuclease activity and will replace the last
added nucleotide. The enzyme will stop DNA synthesis and last mismatched
nucleotide will enter into the proof reading site. It will be hydrolyzed and a
fresh nucleotide will be added Fig. 5.13.

If the tautomeric shift

is slow, DNA
polymerase moves on
and a mismatch
nucleotide is
incorporated into the
DNA.

Fig. 5.13: 3´→5´ exonuclease activity of DNA polymerase I. 107

Block 2 DNA Replication

SAQ 3
State whether the following statement is true of false:

i) DNA cannot afford to have mistakes during DNA replication as it mayl

cause mutations. (True/False)

ii) The chance of making one mistake in E.coli, during DNA replication, is
one for every 109 to 1010 added nucleotides. (True/False)

iii) The bases like adenine and cytosine exist in keto and enol. (True/False)

iv) The bases like guanine and thymine exist in keto and enol form.
(True/False)

v) DNA polymerase I (DNAP I) adds nucleotides in the 3´ to 5´ direction.

(True/False)

5.7 SUMMARY
 The DNA replication produces two identical daughter DNA molecules.

 It is semi-conservative and daughter molecules contain one each of old

strand and one new strand.

 It is bidirectional and begins at sites called origin of replication.

 DNA replication is catalyzed by enzymes called DNA polymerases and

each polymerase requires a short RNA/DNA primer having a free 3´–OH
group as DNA polymerases cannot begin DNA synthesis de novo.

 There is continuous DNA synthesis on the leading strand and

discontinuous synthesis in the form of short pieces called Okazaki
fragments on the lagging strand.

 There is a high degree of accuracy during DNA replication and there are
different mechanisms to maintain fidelity of replication.

5.8 TERMINAL QUESTIONS

1. Explain the chemistry behind phosphodiester bond formation.

2. Explain the three models of DNA replication.

3. Prove experimentally that DNA replication occurs by semi-conservative

model.

4. Discuss the different properties of DNA polymerase I enzyme.

5. Discuss the accuracy rate of DNA replication in prokaryotes. How the

accuracy of replication is achieved?

6. Define leading and lagging strand synthesis.

7. Provide experimental evidence that DNA replication occurs continuously

on the leading strand and discontinuously in the form of short pieces on
the lagging strand.
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Unit 5 Introduction to Replication

5.9 ANSWERS
Self Assessment Questions
1. a) i) False

ii) False

iii) True

iv) True

b) i) DNA replication

ii) Reverse Transcription

2. a) i) The point of separation or unwinding in DNA where

replication begins.

ii) At the origin, two replication forks are formed and move
away from the origin in both the directions as parental double
helix is separated.

iii) Okazaki fragments are short, newly synthesized DNA

fragments that are formed on the lagging strand template
during DNA replication and are 1000 to 2000 nucleotides
long in E. coli and are 100 to 200 nucleotides long
in eukaryotes.

iv) Replication fork is a structure that forms during DNA

replication within the nucleus by the initiation of enzyme
helicase. The resulting structure has two branching "prongs",
each one made up of a single strand of DNA that acts as a
template.

b) i) False

ii) True

iii) True

iv) False

v) True

3. i) True

ii) True

iii) False

iv) True

v) False 109
Block 2 DNA Replication

Terminal Questions

1. During DNA polymerization reaction, free 3´ hydroxyl group of the last

nucleotide at the 3’ end of the growing chain attacks the alpha
phosphorus of the incoming nucleotide thus making a phosphodiester
bond and PPi is released. This reaction is endergonic and
thermodynamically unfavourable. For more details refer section 5.2.2.

2. The three models of DNA replication are conservative, semi-

conservative and dispersive. For more details refer section 5.3.

3. Meselson and Stahl’s experiment. Refer section 5.3.1.

4. DNA polymerase I (DNAP I) adds nucleotides in the 5´ to 3´ direction

one at a time. DNAP I has three activities: 5´→3´ exonuclease activity
which removes RNA primers at the end of each Okazaki fragment,
5´→3´ polymerase activity which fills the gap between Okazaki
fragments, and 3´→5´ exonuclease activity which functions in
proofreading. Refer section 5.6.

5. Refer section 5.6.

6. By proofreading. Refer section 5.5.3.

7. Pulse-chase experiments helped to prove discontinuous DNA synthesis.

For more details refer section 5.5.3.

110