Bth 202 molecular biology

PROF. BALASUBRAMANIAN SATHYAMURTHY 2016 EDITION BTH – 202: MOLECULAR BIOLOGY
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FOR MSC BIOTECHNOLOGY STUDENTS
2014 ONWARDS
Biochemistry scanner
THE IMPRINT
BTH – 202: MOLECULAR BIOLOGY
As per Bangalore University (CBCS) Syllabus
2016 Edition
BY: Prof. Balasubramanian Sathyamurthy
Supported By:
Ayesha Siddiqui
Kiran K.S.
THE MATERIALS FROM “THE IMPRINT (BIOCHEMISTRY SCANNER)” ARE NOT
FOR COMMERCIAL OR BRAND BUILDING. HENCE ONLY ACADEMIC CONTENT
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I dedicate thI dedicate thI dedicate thI dedicate this material to my spiritual guru Shri Raghavendra swamigal,is material to my spiritual guru Shri Raghavendra swamigal,is material to my spiritual guru Shri Raghavendra swamigal,is material to my spiritual guru Shri Raghavendra swamigal,
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AASHITA SINHA ASHWINI BELLATTI
BHARATH K CHAITHRA
GADIPARTHI VAMSEEKRISHNA KALYAN BANERJEE
KAMALA KISHORE
KIRAN KIRAN H.R
KRUTHI PRABAKAR KRUPA S
LATHA M MAMATA
MADHU PRAKASHHA G D MANJUNATH .B.P
NAYAB RASOOL S NAVYA KUCHARLAPATI
NEHA SHARIFF DIVYA DUBEY
NOOR AYESHA M PAYAL BANERJEE
POONAM PANCHAL PRAVEEN
PRAKASH K J M PRADEEP.R
PURSHOTHAM PUPPALA DEEPTHI
RAGHUNATH REDDY V RAMYA S
RAVI RESHMA
RUBY SHA SALMA H.
SHWETHA B S SHILPI CHOUBEY
SOUMOUNDA DAS SURENDRA N
THUMMALA MANOJ UDAYASHRE. B
DEEPIKA SHARMA
EDITION : 2016
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M. SC. BIOTECHNOLOGY – SECOND SEMESTER
BTH – 202: MOLECULAR BIOLOGY
4 units (52 hrs)
UNIT – 1 STRUCTURE AND PROPERTIES OF DNA AND RNA 6 Hrs
Information flow in biological systems: Central dogma. Biochemical evidences for DNA
as genetic material. Watson and Crick model of DNA, different forms of DNA (A, B, Z
DNA), Properties and types of DNA. UV absorption, Denaturation and renaturation,
thermodynamics of melting of the double helix, kinetics of unwinding of the double
helix, Interaction with small ions. Structure and different types of RNA.
Unit – 2 DNA REPLICATION 8 Hrs
Characteristics and functions of bacterial DNA polymerases, Mechanism of Prokaryotic
DNA replication, Models of replications in prokaryotes. Fidelity of replication, Nearest
neighbor frequency analysis. Eukaryotic DNA poymerases and mechanism of
replication. Telomere synthesis – telomereases. Replication of viral DNA, rolling circle
model. Inhibitors of replication.
UNIT – 3 TRANSCRIPTION: 8 Hrs
Characteristics and function of bacterial RNA polymerases, mechanism of transcription
and regulation. Eukaryotic RNA Polymerases – transcription factors, mechanism of
transcription and regulation. Stringenet response. Post transcriptional modifications of
mRNA ( 5’ CAP formation, poly adenylation, spliciosome assembly, splicing, editing),
stability. Processing of t RNA and r RNA. Inhibitors of transcription. Ribozyme
technology: mechanism of action and applications.
UNIT – 4 TRANSLATION: 8 Hrs
Genetic Code, Wobble hypothesis. Ribosome assembly, mechanism of activation of
amino acids. Mechanism of translation in prokaryotes and eukaryotes. Differences
between prokaryotes and eukaryotes protein synthesis, codon usage, inhibitors of
protein synthesis. Co and post translational modifications of proteins. Control of
translation in eukaryotes (Antisense, Heme and interferons).
UNIT – 5 REGULATION OF GENE EXPRESSION: 10 Hrs
Gene regulation, operon model – Inducible and repressible systems, lac, gal, trp, his
and arabinose operon. Attenuation , positive and negative regulation, role of cAMP
and CRP in the expression of lac gene, catabolite repression, regulation of eukaryotic

gene expression, transcription control, cis control elements, promoters, enhancers,
transacting factors, homeobox in the control of developments in insects and
vertebrates. DNA binding motifs of transcription factors, post transcriptional control.
UNIT – 6 PROTEIN LOCALISATION AND TARGETING: 5 Hrs
Export of secretory protein – signal hypothesis, transport and localization of proteins to
mitochondria, chloroplast, peroxysomes and membrane.
UNIT – 7: DNA DAMAGE AND REPAIR: 5 Hrs
DNA damage, alkylation, deamination, oxidation, UV radiation, Repair mechanism –
photo activation, excision repair, post replication repair, mismatch repair and SOS
repair.
UNIT – 8: GENE SILENCING: 2 Hrs
Definition, types – transcriptional and post transcriptional gene silencing – RNAi
pathway (siRNA and miRNA).

UNIT – 1 STRUCTURE AND PROPERTIES OF DNA AND RNA
Information flow in biological systems: Central dogma. Biochemical evidences for
DNA as genetic material. Watson and Crick model of DNA, different forms of DNA
(A, B, Z DNA), Properties and types of DNA. UV absorption, Denaturation and
renaturation, thermodynamics of melting of the double helix, kinetics of
unwinding of the double helix, Interaction with small ions. Structure and different
types of RNA.
INFORMATION FLOW IN BIOLOGICAL SYSTEMS
The central dogma defines the paradigm of molecular biology. Genes are perpetuated as
sequences of nucleic acid, but function by being expressed in the form of proteins.
Replication is responsible for the inheritance of genetic information. Transcription and
translation are responsible for its conversion from one form to another.
FLOW OF INFORMATION: CENTRAL DOGMA OF MOLECULAR BIOLOGY
Below Figure illustrates the roles of replication, transcription, and translation, viewed
from the perspective of the central dogma:
The perpetuation of nucleic acid may involve either DNA or RNA as the genetic material.
Cells use only DNA. Some viruses use RNA, and replication of viral RNA occurs in the
infected cell.
The expression of cellular genetic information usually is unidirectional. Transcription of
DNA generates RNA molecules that can be used further only to generate protein
sequences; generally they cannot be retrieved for use as genetic information.
Translation of RNA into protein is always irreversible.
The central dogma states that information in nucleic acid can be perpetuated or
transferred, but the transfer of information into protein is irreversible.

The genomes of all living organisms consist of duplex DNA. Viruses have genomes that
consist of DNA or RNA; and there are examples of each type that are double-stranded
(ds) or single-stranded (ss). Details of the mechanism used to replicate the nucleic acid
vary among the viral systems, but the principle of replication via synthesis of
complementary strands remains the same, as illustrated in Figure
Double stranded and single stranded nucleic acid both replicate by synthesis of
complementary strands governed by the rules of base pairing
Cellular genomes reproduce DNA by the mechanism of semi-conservative replication.
Double-stranded virus genomes, whether DNA or RNA, also replicate by using the
individual strands of the duplex as templates to synthesize partner strands.
Viruses with single-stranded genomes use the single strand as a template to synthesize
a complementary strand; and this complementary strand in turn is used to synthesize
its complement, which is, of course, identical with the original starting strand.
Replication may involve the formation of stable double-stranded intermediates or may
use doublestranded nucleic acid only as a transient stage
The restriction to unidirectional transfer from DNA to RNA is not absolute. It is
overcome by the retroviruses, whose genomes consist of single-stranded RNA molecules.
During the infective cycle, the RNA is converted by the process of reverse transcription
into a single-stranded DNA, which in turn is converted into a double-stranded DNA.
This duplex DNA becomes part of the genome of the cell, and is inherited like any other
gene. So reverse transcription allows a sequence of RNA to be retrieved and used as
genetic information. The existence of RNA replication and reverse transcription

establishes the general principle that information in the form of either type of nucleic
acid sequence can be converted into the other type.
Throughout the range of organisms, with genomes varying in total content over a
100,000 fold range, a common principle prevails. The DNA codes for all the proteins
that the cell(s) of the organism must synthesize; and the proteins in turn (directly or
indirectly) provide the functions needed for survival.
The nucleic acid codes for the protein(s) needed to package the genome and also for any
functions additional to those provided by the host cell that are needed to reproduce the
virus during its infective cycle.
BIOCHEMICAL EVIDENCES FOR DNA AS GENETIC MATERIAL
AIM:
To prove DNA as the genetic material in the most of living organisms
PRINCIPLE:
We need to discuss this in an historical context. During the 19th century most
scientists thought that a bit of the essence of each and every body part was put into the
sperm and egg and that at conception a blending essences occurred. This theory was
called Blending Inheritance. It was based on a non-rigorous observation of nature.
Complex characteristics were examined and careful counts of the number and type of
progeny were not performed. It is not correct.
By the 1930's, the scientific community had accepted the existence of discrete genetic
elements and that these genetic elements were probably carried on or by chromosomes.
The burning question of the day was what type of molecule carried the genetic
information. During the 1940's it was known that chromosomes contained both DNA
and small basic proteins called histones.
It was also clear that the genetic material:
Must be of sufficient complexity to encode tens of thousands of different proteins each
of which are hundreds to thousands of amino acids long.
Must be able to be replicated with high fidelity each and every cell division so that it
could be passed down to future generations.
Must be very stable, that is it must not be subject to a high rate of randomization.
Some cells in your body survive for 80 years
Must be able to be altered by mutations. Mutations are changes in the genetic
material.

PROCEDURE:-
EXPERIMENT NO: 1
DNA is the genetic material of bacteria(FRED GRIFFITH-1928)
Bacterial transformation provided the first proof that DNA is the : genetic material.
Genetic properties can be transferred from one : bacterial strain to another by
extracting DNA from the first strain : and adding it to the second strain.
The idea that genetic material is nucleic acid had its roots in the discovery of
transformation in 1928.
The bacterium Pneumococcus kills mice by causing pneumonia.
The virulence of the bacterium is determined by its capsular polysaccharide.
This is a component of the surface that allows the bacterium to escape destruction by
the host.
Several types (I, II, III) of Pneumococcus have different capsular polysaccharides. They
have a smooth (S) appearance.
Each of the smooth Pneumococcal types can give rise to variants that fail to produce the
capsular polysaccharide.
These bacteria have a rough (R) surface (consisting of the material that was beneath the
capsular polysaccharide).
They are avirulent. They do not kill the mice, because the absence of the polysaccharide
allows the animal to destroy the bacteria. When smooth bacteria are killed by heat
treatment, they lose their ability to harm the animal.
But inactive heat-killed S bacteria and the ineffectual variant R bacteria together have a
quite different effect from either bacterium by itself. shows that when they are jointly
injected into an animal, the mouse dies as the result of a Pneumococcal infection.
Virulent S bacteria can be recovered from the mouse postmortem.
In this experiment, the dead S bacteria were of type III. The live R bacteria had been
derived from type II. The virulent bacteria recovered from the mixed infection had the
smooth coat of type III.
So some property of the dead type III S bacteria can transform the live R bacteria so that
they make the type III capsular polysaccharide, and as a result become virulent.

Neither heat killed s-type nor live r-type bacteria can kill mice,but simultaneous
infection of them both can kill mice just as effectively as the live s-type
identification of the component of the dead bacteria responsible for transformation. This
was called the transforming principle. It was purified by developing a cell-free system,
in which extracts of the dead S bacteria could be added to the live R bacteria before
injection into the animal. Purification of the transforming principle in 1944 showed that
it is deoxyribonucleic acid (DNA).
EXPERIMENT NO: 2
Oswald Avery, Colin MacLeod and MacLyn McCarty 1944-Identity of the
Transforming Principle
The transforming principle could be isolated as a cell-free extract and was fully active.
The stability of the principle’s transforming activity to heat treatment at 65°C suggested
that it was not a protein (such high temperatures denature most proteins).
In 1944, Oswald Avery, C. M. MacLeod, and M. J. McCarty succeeded in isolating a
highly purified preparation of DNA from the type IIIS bacteria.
The preparation of this type IIIS DNA was fully active as a transforming agent and could
transform type IIR cells into type IIIS cells in a test tube.
If the DNA was destroyed by deoxyribonuclease (an enzyme that specifically attacks
DNA), all transforming activity was lost.
It therefore seemed clear that DNA was “functionally active in determining the
biochemical activities and specific characteristics of pneumococcal cells”.
These experiments by themselves, however, do not establish that DNA is itself the
genetic material. Perhaps DNA acts upon the genetic material of the recipient cell
changing its genes to resemble the genes of the DNA donor? A clear demonstration was
provided by experiments on bacterial viruses.

Conclusion from this experiment:
DNA is the transforming principle.
Avery was as surprised by this as anyone else. When he began this series of
experiments he fully expected that the transforming factor would be a component of the
polysaccharide coat itself
EXPERIMENT.NO:3
DNA is the genetic material of viruses (Alfred Hershey and Martha Chase 1952)
Phage T2 is a virus that infects the bacterium E. coli. When phage particles are added to
bacteria, they adsorb to the outside surface, some material enters the bacterium, and
then -20 minutes later each bacterium bursts open (lyses) to release a large number of
progeny phage.
The results of an experiment in 1952 in which bacteria were infected with T2 phages
that had been radioactively labelled either in their DNA component (with 32P) or in their
protein component (with 35S).
The infected bacteria were agitated in a blender, and two fractions were separated by
centrifugation. One contained the empty phage coats that were released from the
surface of the bacteria. The other fraction consisted of the infected bacteria themselves.
Most of the 32P label was present in the infected bacteria. The progeny phage particles
produced by the infection contained ~30% of the original 32P label. The progeny
received very little—less than1%—of the protein contained in the original phage
population.
The phage coats consist of protein and therefore carried the 35S radioactive label. This
experiment therefore showed directly that only theDNA of the parent phages enters the

bacteria and then becomes part of the progeny phages, exactly the pattern of
inheritance expected of genetic material.
A phage (virus) reproduces by commandeering the machinery of an infected host cell to
manufacture more copies of it. The phage possesses genetic material whose behavior is
analogous to that of cellular genomes: its traits are faithfully reproduced, and they are
subject to the some rules that govern inheritencethe case ti reinforces the general
conclusion that the genetic material is DNA, Whether the part of the genome of a cell or
virus.
CONCLUSION:
These experiments finally convinced the scientific world that DNA and not protein must
be the genetic material. No other interpretation was reasonable. Now people had to face
the fact that a repeating polymer of only 4 different nucleotides was able to encode
every protein that a cell needed to function.
EXPERIMENT.NO:4
DNA is the genetic material of animal cells
When DNA is added to populations of single eukaryotic cells growing in culture, the nucleic acid
enters the cells, and in some of them results in the production of new proteins. When a purified
DNA is used, its incorporation leads to the production of a particular protein.

Although for historical reasons these experiments are described as transfection when
performed with eukaryotic cells, they are a direct counterpart to bacterial
transformation.
The DNA that is introduced into the recipient cell becomes part of its genetic material,
and is inherited in the same way as any other part.
Its expression confers a new trait upon the cells (synthesis of thymidine kinase in the
example of the figure). At first, these experiments were successful only with individual
cells adapted to grow in a culture medium.
Since then, however, DNA has been introduced into mouse eggs by microinjection; and
it may become a stable part of the genetic material of the mouse
Such experiments show directly not only that DNA is the genetic material in eukaryotes,
but also that it can be transferred between different species and yet remain functional.
The genetic material of all known organisms and many viruses is DNA. However, some
viruses use an alternative type of nucleic acid, ribonucleic acid (RNA), as the genetic
material. The general principle of the nature of the genetic material, then, is that it is
always nucleic acid; in fact, it is DNA except in the RNA viruses.
WATSON AND CRICK MODEL OF DNA
The salient features of the Watson-Crick model for the commonly found DNA ( B-DNA)
are:
1. DNA molecule consists of two helical polynucleotide chains which are coiled around (or
wrapped about) a common axis in the form of a right handed double helix. The two

helices are wound in such a way so as to produce 2 interchain spacings or grooves, a
major or wide groove (width 12 Å, depth 8.5 Å) and a minor or narrow groove (width 6 Å,
depth 7.5 Å).
2. The two grooves arise because the glycosidic bonds of a base pair are not diametrically
opposite each other.
3. The minor groove contains the pyrimidine O-2 and the purine N-3 of the base pair,
and the major groove is on the opposite side of the pair.
4. Each groove is lined by potential hydrogen bond donor and acceptor atoms.
5. The two helices wind along the molecule parallel to the phosphodiester backbones.
6. The phosphate and deoxyribose units are found on the periphery of the helix, whereas
the purine and pyrimidine bases occur in the centre.
7. The planes of the bases are perpendicular to the helix axis.
8. The planes of the sugars are almost at right angles to those of the bases.
9. The diameter of the helix is 20 Å. The bases are 3.4 Å apart along the helix axis and are
related by a rotation of 36 degrees. Therefore, the helical structure repeats after 10
residues on each chain, i.e., at intervals of 34 Å. In other words, each turn of the helix
contains 10 nucleotide residues.
10. The two chains are held together by hydrogen bonds between pairs of bases.
11. Adenine always pairs with thymine by 2 hydrogen bonds and guanine with cytosine
with 3 hydrogen bonds. This specific positioning of the bases is called base
complementarity.
12. The individual hydrogen bonds are weak in nature but, a large number of them involved
in the DNA molecule confer stability to it. It is now thought that the stability of the DNA
molecule is primarily a consequence of van der Waals forces between the planes of
stacked bases.
13. Base complementarity of the polynucleotide chain.
14. An important feature of the double helix is the specificity of the pairing of bases. Pairing
always occurs between adenine and thymine and between guanine and cytosine.
Steric factor:
The steric restriction is imposed by the regular helical nature of the sugar-phosphate
backbone of each polynucleotide chain.
Hydrogen-bonding factor:

The base pairing is further restricted by hydrogen-bonding requirements. The hydrogen
atoms in purine and pyrimidine bases have well-defined positions. Adenine cannot pair
with cytosine because there would be two hydrogen atoms near one of the bonding
positions and none at the other. Similarly, guanine cannot pair with thymine.
DIFFERENT FORMS OF DNA (A, B, Z DNA)
Characteristics A-DNA B-DNA C-DNA Z-DNA
Conditions 75% relative
humidity
Na+, K+, Cs+
ions
92% relative
humidity
Low ion
strength
60%
relative
humidity
Li+ ions
Very high salt
concentration
Shape Broadest Intermediate Narrow Narrowest
Helix sense Right-handed Right-handed Right-
handed
Left-handed
Helix diameter 25.5 Å 23.7 Å 19.0 Å 18.4 Å
Rise per base pair
(‘h’)
2.3 Å 3.4 Ã 3.32 Å 3.8 Å
Base pairs/helix
turn (‘n’)
11 10.4 9.33 12 (= 6 dimers)
Helix pitch (h × n) 25.30 Å 35.36 Å 30.97 Å 45.60 Å
Rotation / base pair + 32.72° + 34.61° + 38.58° –60° (per
dimer)
Base pair tilt 19° 1° 7.8° 9°

Glycosidic bond anti anti — anti for C, T
syn for A, G
Major groove Narrow and
very deep
Wide and
quite deep
— Flat
Minor groove Very broad
and shallow
Narrow and
quite deep
— Very narrow
and deep
Structure —
PROPERTIES AND TYPES OF DNA
BASE PAIRING
The double helix model for DNA was given by Watson and Crick in 1953. According to
it, the two polynucleotide chains in the double helix associate by hydrogen bonding
between the nitrogenous bases. G can hydrogen bond specifically only with C, while A
can bond specifically only with T. These reactions are described as base pairing, and
the paired bases (G with C, or A with T) are said to be complementary.
The model proposed that the two polynucleotide chains run in opposite directions
known as antiparallel arrangement.
One strand runs in the 5'—>3' direction, while its partner runs 3'—»5'.
Pairing always occurs between adenine and thymine and between guanine and cytosine.
Base-pairing is due to steric and hydrogen-bonding factors.
Steric factor:
The glycosidic bonds that are attached to a base pair are always 10.85 Å apart. A
purine-pyrimidine base pair fits perfectly in this space. If the base order in one strand is
known, the sequence of bases in the other strand can be predicted.

Hydrogen-bonding factor.
The base pairing is further restricted by hydrogen-bonding requirements. The hydrogen
atoms in purine and pyrimidine bases have well-defined positions. Adenine forms 2
hydrogen bonds with thymine whereas guanine forms 3 with cytosine.
Thus, the G-C bond is stronger by 50% than the A-T bond. The higher The G-C content
of a DNA moelcule, the greater is its buoyant density.
Base Pairing In Watson and Crick Model of DNA
UV ABSORPTION
Changes involved during denaturation:
Increase in absorption of ultraviolet light (= Hyperchromic effect):
As a result of resonance, all of the bases in nucleic acids absorb ultraviolet light. And
all nucleic acids are characterized by a maximum absorption of UV light at wavelengths
near 260 nm. When the native DNA (which has base pairs stacked similar to a stack of

coins) is denatured, there occurs a marked increase in optical absorbancy of UV light by
pyrimidine and purine bases, an effect called hyperchromicity or hyperchromism
whch is due to unstacking of the base pairs. This change reflects a decrease in
hydrogen-bonding.
DENATURATION
Denaturation of DNA is a loss of biologic activity and is accompanied by cleavage of
hydrogen bonds holding the complementary sequences of nucleotides together. This
results in a separation of the double helix into the two constituent polynucleotide
chains. In it, the firm, helical, two stranded native structure of DNA is converted to a
flexible, single-stranded denatured state. The transition from native to a denatured
form is usually very abrupt and is accelerated by reagents such as urea and
formamide, which enhance the aqueous solubility of the purine and pyrimidine groups.
Decrease in specific optical rotation:
Native DNA exhibits a strong positive rotation which is highly decreased upon
denaturation.
Decrease in viscosity:
The solutions of native DNA possess a high viscosity because of the relatively rigid
double helical structure and long, rodlike character of DNA. Disruption of the hydrogen
bonds causes a marked decrease in viscosity.
DNA denaturation curve
EFFECT OF pH AND TEMPERATURE ON DENATURATION:
pH :
Denaturation of DNA helix also occurs at acidic and alkaline
pH values at which ionic changes of the substituents on the purine and pyrimidine
bases can occur. In acid solutions near pH 2 to 3, at which amino groups bind protons,

the DNA helix is disrupted. Similarly, in alkaline solutions near pH 12, the enolic
hydroxyl groups ionize, thus preventing the keto-amino group hydrogen bonding.
Temperature:
The DNA double helix, although stabilized by hydrogen bonding, can be denatured by
heat by adding acid or alkali to ionize its bases. The unwinding of the double helix is
called melting because it occurs abruptly at a certain characteristic temperature called
denaturation temperature or melting temperature (Tm).
Tm : The melting temperature is defined as the temperature at which half the helical
structure is lost.
The melting of DNA is readily monitored by measuring its absorbance of light at
wavelength near 260 nm. The abruptness of the transition indicates that the DNA
double helix is highly cooperative structure, held together by many reinforcing bonds ;
it is stabilized by the stacking of bases as well as by pairing.
Tm can be lowered by the addition of urea which disrupts hydrogen bonds. DNA can be
completely denatured (i.e., separated into a single-stranded structure) by 95%
formamide at room temperature only.

Since the G-C base pair has 3 hydrogen bonds as compared to 2 for A-T, it follows that
DNAs with high concentrations of G and C might be more stable and have a higher Tm
than those with high concentrations of A and T.
DNA melting curves
The Tm is about 72°C for Escherichia coli DNA (50% G-C pairs) and 79°C for the
bacterium, Pseudomonas aeruginosa DNA (66% G-C pairs).
RENATURATION:
If a solution of denatured DNA, prepared by heating, is cooled slowly to room
temperature, some amount of DNA is renatured.
Maximum reversibility (50-60%) is usually attained by annealing i.e. slow cooling the
denatured DNA, i.e., holding the solution at a temperature about 25°C below Tm and
above a concentration of 0.4 M Na+ for several hours.

The restoration occurs because the complementary bases reunite by hydrogen bonds
and the double helix again forms.
Reannealing
Steps in the denaturation and renaturation of DNA fragments:
I. Nucleation reaction: In this hydrogen bonds form between two complementary single
strands; this is a bimolecular, second-order reaction.
II. Zippering reaction: In this hydrogen bonds form between all the bases in the
complementary strands; this is a unimolecular, first-order reaction.
THERMODYNAMICS OF MELTING OF THE DOUBLE HELIX
Nucleic acid thermodynamics is the study of how temperature affects the nucleic acid
structure of double-stranded DNA (dsDNA). The melting temperature (Tm) is defined as
the temperature at which half of the DNA strands are in the random coil or single-
stranded (ssDNA) state. Tm depends on the length of the DNA molecule and its
specific nucleotide sequence. DNA, when in a state where its two strands are
dissociated (i.e., the dsDNA molecule exists as two independent strands), is referred to
as having been denatured by the high temperature.
KINETICS OF UNWINDING OF THE DOUBLE HELIX
Several formulas are used to calculate Tm values. Some formulas are more accurate in
predicting melting temperatures of DNA duplexes .For DNA oligonucleotides, i.e. short
sequences of DNA, the thermodynamics of hybridization can be accurately described as
a two-state process. In this approximation one neglects to possibility of intermediate
partial binding states in the formation of a double strand state from two single stranded
oligonucleotides. Under this assumption one can elegantly describe the thermodynamic

parameters for forming double-stranded nucleic acid AB from single-stranded nucleic
acids A and B.
AB ↔ A + B
The equilibrium constant for this reaction is
.
According to the Van´t Hoff equation, the relation between free energy, ∆G, and K is
∆G° = -RTln K, where R is the ideal gas law constant, and T is the kelvin temperature of
the reaction. This gives, for the nucleic acid system,
.
The melting temperature, Tm, occurs when half of the double-stranded nucleic acid has
dissociated. If no additional nucleic acids are present, then [A], [B], and [AB] will be
equal, and equal to half the initial concentration of double-stranded nucleic acid,
[AB]initial. This gives an expression for the melting point of a nucleic acid duplex of
.
Because ∆G° = ∆H° -T∆S°, Tm is also given by
.
The terms ∆H° and ∆S° are usually given for the association and not the dissociation
reaction (see the nearest-neighbor method for example). This formula then turns into:
,
where [B]total < [A]total.
As mentioned, this equation is based on the assumption that only two states are
involved in melting: the double stranded state and the random-coil state. However,
nucleic acids may melt via several intermediate states. To account for such complicated
behavior, the methods of statistical mechanics must be used, which is especially
relevant for long sequences.
From the observation of melting temperatures one can experimentally determine the
thermodynamic parameters. Vice versa, and important for applications, when the

thermodynamic parameters of a given nucleic acid sequence are known, the melting
temperature can be predicted. It turns out that for oligonucleotides, these parameters
can be well approximated by the nearest-neighbor model.
INTERACTION WITH SMALL IONS
The structure and dynamics of the grooves of DNA are of immense importance for
recognition of DNA by proteins and small molecules as well as for the packaging of DNA
into nucleosomes and viral particles.
Although there is general agreement that the minor groove of DNA varies in a sequence-
dependent manner and is narrow in AT regions, alternative models have been presented
to explain the molecular basis for the groove narrowing. In one model the groove
narrowing results from direct, short-range interactions among DNA bases. In this model
the minor groove width of a given sequence is fixed, and any localization of monovalent
cations in the groove does not affect the groove structure. In an alternative model the
narrow minor groove of A-tracts is proposed to originate from sequence-dependent
localization of water and cations.
Ion dynamics and exchange make experimental tests of these models difficult, but they
can be directly tested by determining how DNA minor-groove structure responds to
cation positions in the course of molecular dynamics (MD) simulations.
To carry out such a test, we have conducted a long MD simulation on the sequence
d(CGCGAATTCGCG)2 in the presence of ions and water. We have analyzed the major
structures that exist and the correlation between ion population and minor groove
width. The results clearly show a time-dependent influence of ion positions on minor
groove structure. When no ions interact with the groove, the groove is wide. Ion-water
interactions narrow the groove through two distinct interactions: (i) ions interact
directly with the DNA bases in the minor groove, such as cross-strand thymine oxygens
(O2) in the sequence above, to give an internal ion-spine of hydration, or (ii) ions interact
with phosphate groups in the AT sequence while water molecules in the minor groove
interact directly with the bases. Some variations on these limiting models are possible
in a dynamic DNA-water-ion structure, but it is clear that ion and water interactions at
AT base pair sequence sites are required to yield the observed narrow minor groove in
AT sequences.

STRUCTURE AND DIFFERENT TYPES OF RNA.
Ribonucleic acid (RNA), like DNA, is a long, unbranched macromolecule consisting of
nucleotides joined by 3’ to 5’ phosphodiester bonds. The number of ribonucleotides in
RNA ranges from as few as 75 to many thousands.
Types of RNA
In all procaryotic and eucaryotic organisms, 3 general types of RNAs are found:
ribosomal, transfer and messenger RNAs. Each of these polymeric forms serves as
extremely important informational links between DNA, the master carrier of information
and proteins. The 3 types of RNA molecules differ from each other by size, function and
general stability.
Ribosomal RNA (rRNA) or Insoluble RNA:
It is the most stable form of RNA and is found in ribosomes. It has the highest molecular
weight and is sedimented when a cell homogenate containing 10−2 M of Mg2+ is
centrifuged at high speed (100,000 gravity for 120 minutes).
In the bacterium, Escherichia coli, there are 3 kinds of RNA called 23 s, 16 s, and 5 s
RNA because of sedimentation behaviour.
These have molecular weights of 1,200,000, 550,000 and 36,000 respectively.
One molecule of each of these 3 types of rRNA is present in each ribosome. Ribosomal
RNA is most abundant of all types of RNAs and makes up about 80% of the total RNA of
a cell.
Ribosomal RNA represents about 40-60% of the total weight of ribosomes.
Ribosomes rRNA
Procaryotic ribosomes
30 s
50 s
16 s
5 s, 23 s
Eucaryotic ribosomes

40 s
60 s
18 s
5 s, 28 s
rRNA has G-C contents more than 50%. The rRNA molecule appears as a single
unbranched polynucleotide strand (= primary structure). At low ionic strength, the
molecule shows a compact rod with random coiling. But at high ionic strength, the
molecule reveals the presence of compact helical regions with complementary base
pairing and looped outer region ( = secondary structure).
The helical structure results from a folding back of a single-stranded polymer at areas
where hydrogen bonding is possible because of short lengths of complementary
structures. The double helical secondary structures in RNA can form within a single
RNA molecule or between 2 separate RNA molecules. RNAs can often assume even more
complex shapes as in bacteria.
Transfer RNA (tRNA) or Soluble RNA (sRNA):
Transfer RNA is the smallest polymeric form of RNA. These molecules seem to be
generated by the nuclear processing of a precursor molecule. In abundance, the tRNA
comes next to rRNA and amounts to about 15% of the total RNA of the cell. The tRNA
remains dissolved in solution after centrifuging a broken cell suspension at 100,000 X
gravity for several hours. The tRNA molecules serve a number of functions, the most
important of which is to act as specific carriers of activated amino acids to specific sites
on the protein- synthesizing templates.
Common structural features of tRNAs
All tRNA molecules have a common design and consist of 3 folds giving it a shape of the
cloverleaf with four arms; the longer tRNAs have a short fifth or extra arm. The actual
3-dimensional structure of a tRNA looks more like a twisted L than a cloverleaf
All tRNA molecules are unbranched chains containing from 73 to 93 ribonucleotide
residues, corresponding to molecular weights between 24,000 and 31,000
They contain from 7 to 15 unusual modified bases. Many of these unusual bases are
methylated or dimethylated derivatives of A, U, G and C.
Methylation prevents the formation of certain base pairs so that some of the bases
become accessible for other interactions. Methylation imparts hydrophobic character to
some portions of tRNA molecules which may be important for their interaction with the
synthetases and with ribosomal proteins.

The 5’ end of tRNAs is phosphorylated. The 5’ terminal residue is usually guanylate
(pG).
The base sequence at the 3’ end of all tRNAs is CCA. All amino acids bind to this
terminal adenosine via the 3’-OH group of its ribose.
50% of the nucleotides in tRNAs are base-paired to form double helices.
5 groups of bases which are not base-paired. These 5 groups, of which 4 form ‘loops’,
are :
The 3′ CCA terminal region,
The ribothymine-pseudouracil-cytosine ( = T φ C) loop,
The ‘extra arm’ or little loop, which contains a variable number of residues,
The dihydrouracil ( = DHU) loop, which contains several dihydrouracil residues, and
The anticodon loop, which consists of 7 bases with the sequence, 5′ — pyrimidine —
pyrimidine —X —Y—Z — modified purine — variable base — 3′
The 4 loops are recognition sites. Each tRNA must have at least two such recognition
sites : one for the activated amino acid-enzyme complex with which it must react to
form the aminoacyl-tRNA and another for the site on a messenger RNA molecule which
contains the code (codon) for that particular amino acid.
A unique similarity among all tRNA molecules is that the overall distance from CCA at
one end to the anticodon at the other end is constant. The difference in nucleotide

numbers in various tRNA molecules is, in fact, compensated for by the size of the “extra
arm”, which is located between the anticodon loop and TΨ C loop.
Messenger RNA (mRNA) or Template RNA
Messenger RNA is most heterogeneous in size and stability
among all the types of RNAs. It has large molecular weight
approaching 2 × 106 and amounts to about 5% of the total
RNA of a cell. It is synthesized on the surface of DNA
template. Thus, it has base sequence complementary to
DNA and carries genetic information or ‘message’ (hence its
nomenclature) for the assembly of amino acids from DNA to
ribosomes, the site of protein synthesis.
In procaryotic cells, mRNA is metabolically unstable with a high turnover rate whereas
it is rather stable in eucaryotes. It is synthesized by DNA-dependent RNA polymerase.
On account of its heterogeneity, mRNA varies greatly in chain length. Since few proteins
contain less than 100 amino acids, the mRNA coding for these proteins must have at
least 100 × 3 or 300 nucleotide residues.
In E. coli, the average size of mRNA is 900 to 1,500 nucleotide units. If mRNA carries
the codes for the synthesis of simple protein molecule, it is called monocistronic type
and if it codes for more than one kind of protein, it is known as polycistronic type as in
Escherichia coli.
The mRNAs are single-stranded and complementary to the sense strand of their
respective structural genes. Although both types of mRNA molecules (prokaryotic and
eukaryotic) are synthesized with a triphosphate group at the 5′ end, there is a basic
difference between the two the eukaryotic mRNA molecules, especially those of
mammals, have some peculiar characteristics. The 5’ end of mRNA is ‘capped’ by a 7-
methylguanosine triphosphate which is linked to an adjacent 2’- O-methylribonucleo
side at its 5’-hydroxyl through the 3 phosphates The other end of most mRNA
molecules, the 3’ hydroxyl end, has attached a polymer of adenylate residues, 20–250
nucleotides in length.

Unit – 2 DNA REPLICATION
Characteristics and functions of bacterial DNA polymerases, Mechanism of
Prokaryotic DNA replication, Models of replications in prokaryotes. Fidelity of
replication, Nearest neighbor frequency analysis. Eukaryotic DNA poymerases and
mechanism of replication. Telomere synthesis – telomereases. Replication of viral
DNA, rolling circle model. Inhibitors of replication.
CHARACTERISTICS AND FUNCTIONS OF BACTERIAL DNA POLYMERASES ENZYMES
INVOLVED AT DIFFERENT STEPS OF REPLICATION
DNA POLYMERASES OF E-COLI:
Properties DNA polymerase I and III:

These 2 have fundamental properties that carry critical implications for DNA
replication. All polymerases synthesize DNA only 3’ to 5’ direction, adding a dNTPs to
the 3’ hydroxyl group of a growing chain.
DNA polymerases can add a new deoxyribonucleotide only to a preformed primer strand
that is hydrogen bonded to the template; they are not able to initiate DNA synthesis de
novo by catalyzing the polymerization of free dNTPs.In this respect, DNA polymerases
differ from RNA polymerases, which can initiate the synthesis of new strand of RNA in
the absence of primer.
Mechanism of DNA polymerase I and III:
Introduction:
The E.coli genome encodes three DNA polymerases(DNA polymerase I, II and III or Pol I,
II, III.
DNA polymerase I or Pol I:
This was discovered by Nobel Laurate Arthur Korenberg in E-coli in 1957 and also
called as Kornberg enzyme.
It is a single polypeptide with molecular weight of 109 KDa.
There are about 400 molecules of enzymes in a single bacterial cell
These are roughly spherical in nature with diameter of 6.5 nm and are metallo enzyme
that contains Zn2+
The pol –I enzyme do not execute the DNA synthesis rather, it can concentrate on proof
reading and DNA repair.
The enzyme has following biological functions:

5’ to 3’ Exonuclease activity:
This activity is in the smaller fragment of DNA pol
This activity is responsible to remove the primer from the 5’ end of newly synthesized
chain.
It also plays important role in DNA repair mechanism.
Thymic dimer occurs in DNA, when cell is exposed to ultraviolet light and such dimers
interferes with the movement of replication fork and blocks replication.
Therefore, the 5’ to 3’ exonuclease activity of pol-I can correct such DNA damages by
excession of pyrimidine dimer regions. b. 3’ to 5’ Exonuclease activity:
It involves the elimination of mismatch base pair on primer thus it functions as a proof
reading enzyme.
The ligase subunit of polymerase I known as klenow fragment has this activity
This mismatch base pair results (mol.wt=68 KDa) resulted during polymerization are
corrected by 3’ to 5’ exonuclease activity.
5’ to 3’ Exonuclease activity:
The activity of this enzyme helps in the synthesis of small fragment of DNA and thus
takes part in repair synthesis.
This helps in filling of gaps resulted due to removal of RNA primers.
Klenow fragment:
DNA polymerase I, is not the primary enzyme of replication; instead it performs a host
of clean-up functions during replication, recombination, and repair. The polymerase’s
special functions are enhanced by its 5’→3’ exonuclease activity.
This activity, distinct from the 3’→5’ proofreading exonuclease is located in a structural
domain that can be separated from the enzyme by mild protease treatment. When the
5’→3’exonuclease domain is removed, the remaining fragment (Mr 68,000), the large
fragment called Klenow fragment retains the polymerization and proofreading
activities. The 5’→3’ exonuclease activity of intact DNA polymerase I can replace a
segment of DNA (or RNA) paired to the template strand, in a process known as nick
translation
DNA polymerase III:
It is also known as replicase and is chiefly involved in DNA synthesis in 5’ to 3’ direction
It is the principle replication DNA pol of E.COLI

This enzyme in its action form is associated with 9 proteins to form a
Holoenzymehaving mol.wt.140KDa.
The smallest aggregate of subunits having enzyme activity is known as” Core enzyme”.
It has both 5’ to 3’ polymerization activity and 3’to5’exonucleaseactivity.
MECHANISM OF PROKARYOTIC DNA REPLICATION
INITIATION:
The synthesis of a DNA molecule can be divided into three stages: initiation, elongation,
and termination, distinguished both by the reactions taking place and by the enzymes
required.
ori-c plays important role in initiation of replication.
ORIGIN OF REPLICATION
Ori-C
The E. coli replication origin, oriC, consists of 245 bp; it bears DNA sequence elements
that are highly conserved among bacterial replication origins.
The key sequences of interest here are two series of short repeats: three repeats of a 13
bp sequence and four repeats of a 9 bp sequence.
Arrangement of sequences in the E. coli replication origin, oriC.
Although the repeated sequences (shaded in color) are not identical, certain nucleotides
are particularly common in each position, forming a consensus sequence.
In positions where there is no consensus, N represents any of the four nucleotides. The
arrows indicate the orientations of the nucleotide sequences.
The timing of replication initiation is affected by DNA methylation and interactions with
the bacterial plasma membrane.
The oriC DNA is methylated by the Dam methylase , which methylates the N6 position
of adenine within the palindromic sequence (5’)GATC.
The oriC region of E. coli is highly enriched in GATC sequences—it has 11 of them in its
245 bp, whereas the average frequency of GATC in the E. coli chromosome as a whole is
1 in 256 bp.

Immediately after replication, the DNA is hemimethylated: the parent strands have
methylated oriC sequences but the newly synthesized strands do not.
The hemimethylated oriC sequences are now sequestered for a period by interaction
with the plasma membrane.
After a time, oriC is released from the plasma membrane, and it must be fully
methylated by Dam methylase before it can again bind DnaA. Regulation of initiation
also involves the slow hydrolysis of ATP by DnaA protein, which cycles the protein
between active (with bound ATP) and inactive (with bound ADP) forms on a timescale of
20 to 40 minutes.

At least nine different enzymes or proteins participate in the initiation phase of
replication. They open the DNA helix at the origin and establish a prepriming complex
for subsequent reactions.
DnaA protein
The crucial component in the initiation process is the DnaA protein.
A single complex of four to five DnaA protein molecules binds to the four 9 bp repeats in
the origin, then recognizes and successively denatures the DNA in the region of the
three 13 bp repeats, which are rich in A=T pairs.
This process requires ATP and the bacterial histone like protein HU.
After this other proteins comes into picture and continues the process.
About 20 DnaA protein molecules, each with a bound ATP, bind at the four 9 bp
repeats. The DNA is wrapped around this complex.
The three AUT-rich 13 bp repeats are denatured sequentially.
Hexamers of the DnaB protein bind to each strand, with the aid of DnaC protein.
The DnaB helicase activity further unwinds the DNA in preparation for priming and
DNA synthesis.
ELONGATION
Leading and lagging strand:
• A replication fork (Growing point) is the point at which strands of parental duplex
DNA are separated so that replication can proceed.
• A complex of proteins including DNA polymerase is found at the fork.
• When the circular DNA chromosomeof E. coli is copied, replication begins at a single
point, theorigin. Synthesis occurs at the replication fork, the place atwhich the DNA
helix is unwound and individual strands are replicated.
• Two replication forks move outward from the origin untilthey have copied the whole
replicon, that portion of the genome that contains an origin and is replicated as a unit.
When the replicationforks move around the circle, a structure shaped like theGreek
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.
• In both bacteria and mammals replication forks originate at a structure called a
replication bubble,a local region where the two strands of the parental DNA helix have
been separated from eachother to serve as templates for DNA synthesis

• Events occring
• During replication the DNA double helix must be unwound togenerate separate single
strands. Helicaseswhich binds to atrich region of DNA called replication origins, are
responsible for DNA unwinding. These enzymes useenergy from ATP to unwind short
stretches of helix just ahead of thereplication fork. Once the strands have separated,
they are kept single through specific binding with single-stranded DNA
bindingproteins (SSBs)
• Rapid unwinding can lead to tension and formation of supercoils or supertwists in the
helix. The tension generated by unwinding is relieved, and the unwinding process is
promoted by enzymes known as topoisomerases.
• DNA gyrase is an E. coli topoisomerase that removes the supertwists produced during
replication.
• DNA is probably replicated continuously by DNA polymerase III when the leading strand
is copied. Lagging strand replication is discontinuous, and the fragments are
synthesized in the 5′ to 3′ direction just as in leading strand synthesis.
• First, a special RNA polymerase called a primase synthesizes a short RNA primer,
usually around 10 nucleotides long, complementary to the DNA. It appears that the
primase requires the assistance of several other proteins, and the complex of the
primase with its accessory proteins is called the primosome.
• DNA polymerase III holoenzyme then synthesizes complementary DNA beginning at the
3′ end of the RNA primer.
• In order for DNA polymerases to move along and copy a duplex DNA, helicase must
sequentially unwind the duplexand topoisomerase must remove the supercoils that
form.
• A major complication in the operation of a DNA replicationfork arises from two
properties: the two strands of theparental DNA duplex are antiparallel, and DNA
polymerases (like RNA polymerases) can add nucleotides to thegrowing new strands
only in the 5’→3’ direction.
• Synthesisof one daughter strand, called the leading strand, can proceedcontinuously
from a single RNA primer in the 5’→3’direction, the same direction as movement of the
replicationfork. The problem comes in synthesis of theother daughter strand, called the
lagging strand.

• A cell accomplishes lagging strand synthesis by synthesizing a new primer every few
hundred bases or so on the second parental strand, as more of the strand is exposed by
unwinding. Each of these primers, base-paired to their template strand, is elongated in
the 5’→3’ direction, forming discontinuous segments called Okazaki fragments.
Ligation or Nick translation:
The 5’ to 3’ exonuclease activity at a single strand break (a nick) can occur
simultaneously with polymerization. That is as a, 5’-P nucleotide is removed, a
replacement can be made by the polymerizing activity. Since pol I cannot form a bond
between a 3’-OH group and 5’-monophosphate, the nick moves along the DNA molecule
in the direction of synthesis. This movement is called Nick Translation.
The process steps have followingly:
In this process, an RNA or DNA strand paired to a DNA template is simultaneously
degraded by the 5’ to 3’ exonuclease activity of DNA polymerase I and replaced by the
polymerase activity of the same enzyme.
These activities have a role in both DNA repair and the removal of RNA primers during
replication (both described later).
The strand of nucleic acid to be removed (either DNA or RNA) is shown in green, the
replacement strand in red. DNA synthesis begins at a nick (a broken phosphodiester
bond, leaving a free 3’ hydroxyl and a free 5’ phosphate).
Polymerase I extends the nontemplate DNA strand and moves the nick along the DNA—
a process called nick translation.

A nick remains where DNA polymerase I dissociates, and is later sealed by another
enzyme.
TERMINATION OF REPLICATION
• DNA replication stops when the polymerase complex reaches a termination site on
the DNA in E. coli. The Tus protein binds to these Tersites and halts replication. In
many procaryotes, replication stops randomly when the forks meet.
• Eventually, the two replication forks of the circular E. coli chromosome meet at a
terminus region containing multiple copies of a 20 bp sequence called Ter (for
terminus). The Ter sequences are arranged on the chromosome to create a sort of
trap that a replication fork can enter but cannot leave. The Ter sequences function as
binding sites for a protein called Tus (terminus utilization substance). The Tus-Ter
complex can arrest a replication fork from only one direction.

• Only one Tus-Ter complex functions per replication cycle—the complex first
encountered by either replication fork. Given that opposing replication forksgenerally
halt when they collide, Ter sequences do notseem essential, but they may prevent
overreplication byone replication fork in the event that the other is delayedor halted
by an encounter with DNA damage orsome other obstacle.
• When either replication fork encounters a functional Tus-Ter complex, it halts; the
other fork halts when it meets the first (arrested) fork.
• The final few hundred base pairs of DNA between these large protein complexes are
then replicated (by an as yet unknown mechanism), completing two topologically
interlinked (catenated) circular chromosomes. DNA circles linked in this way are
known as catenanes. Separation of the catenated circles in E. coli requires
topoisomerase IV (a type II topoisomerase). The separated chromosomes then
segregate into daughter cells at cell division. The terminal phase of replication of
other circular chromosomes, including many of the DNA viruses that infect
eukaryotic cells, is similar.
MODELS OF REPLICATIONS IN PROKARYOTES
ASYMMETRIC REPLICATION LOOPED
Mitochondrial DNA replication:
• The origins of replicons in both prokaryotic and eukaryotic chromosomes are static
structures: they comprise sequences of DNA that are recognized in duplex form and

used to initiate replication at the appropriate time. Initiation requires separating the
DNA strands and commencing bidirectional DNA synthesis. A different type of
arrangement is found in mitochondria.
• Replication starts at a specific origin in the circular duplex DNA.But initially only one
of the two parental strands (the H strand in mammalianmitochondrial DNA) is used
as a template for synthesis of a newstrand.
• Synthesis proceeds for only a short distance, displacing the originalpartner (L)
strand, which remains single-stranded. The condition of this region gives rise to its
name as thedisplacement or D loop.
• DNA polymerases cannot initiate synthesis, but require a priming 3'end.
Replicationat the H strand origin is initiated when RNA polymerase transcribes
aprimer. 3' ends are generated in the primer by an endonuclease thatcleaves the
DNA-RNA hybrid at several discrete sites.
• The endonucleaseis specific for the triple structure of DNA-RNA hybrid plus the
displacedDNA single strand. The 3' end is then extended into DNA by theDNA
polymerase.
• A single D loop is found as an opening of 500-600 bases in mammalian
mitochondria. The short strand that maintains the D loop is unstableand turns over;
it is frequently degraded and resynthesized tomaintain the opening of the duplex at
this site.
• Some mitochondrialDNAs possess several D loops, reflecting the use of multiple
origins. The same mechanism is employed in chloroplast DNA, where (inhigher
plants) there are two D loops.
• To replicate mammalian mitochondrial DNA, the short strand in theD loop is
extended. The displaced region of the original L strand becomeslonger, expanding the
D loop.
• This expansion continues until itreaches a point about two-thirds of the way around
the circle. Replicationof this region exposes an origin in the displaced L strand.
Synthesisof an H strand initiates at this site, which is used by a special primasethat
synthesizes a short RNA.
• The RNA is then extended by DNA polymerase,proceeding around the displaced
single-stranded L template inthe opposite direction from L-strand synthesis.

• Because of the lag in its start, H-strand synthesis hasproceeded only a third of the
way around the circle whenL-strand synthesis finishes.
• This releases one completedduplex circle and one gapped circle, which remains
partiallysingle-stranded until synthesis of the H strand iscompleted. Finally, the new
strands are sealed to becomecovalently intact.
• The existence of D loops exposes a general principle.An origin can he a sequence of
DNA that serves to initiateDNA synthesis using one strand as template.
• Theopening of the duplex does not necessarily lead to theinitiation of replication on
the other strand. In the case ofmitochondrial DNA replication, the origins for
replicatingthe complementary strands lie at different locations. Origins thatsponsor
replication of only one strand are also found in the rolling circlemode of replication
SEMICONSERVATIVE REPLICATION
Definition

Each DNA strand serves as a template for the synthesis of a new strand,producing two
new DNA molecules, each with one new strand and one old strand. This is
semiconservativereplication.
Processing
In the semiconservative mode, first proposed by Watson and crick each parental DNA
strand serves as a tempelate for one new or daughter strand and as each new strand is
formed, it is hydrogen- bonded to its parent tempelate.
Thus, replication proceeds, the parental double helix unwinds and then rewinds again
into two new double helices, each of which contains one originally parental strand and
newly formed daughter strand.
Experimental proof: Meselson- Stahl experiment
Aim:
To prove that DNA replication of double stranded DNA follows semiconservative mode of
replication.
Principle:
If the parental DNA "heavy,, density label because the organism has been grown in
medium containing a suitable isotope such as 15N, its strands can be distinguished
from those that are synthesized when the organism is transferred to a medium
containing normal "light" isotopes e.g. 14N. When DNA was extracted from bacteria and
its density measured by centrifugation, the DNA formed bands corresponding to its
density depicting the amount of parental and newly synthesized DNA during the
process of replication.
Procedure:
A simple method was developed by the scientists by which the parental and daughter
strands could be distinguished.
Culture of bacteria ( E. coli ) was grown for many generations in growth medium
containing 15N- labeled NH4Cl as sole source of nitrogen ( called a heavy medium ).
In this way parent DNA was labeled with heavy isotope 15N therebyincreasing the
density of the DNA.
The cells were transferred to a medium containing common isotope of nitrogen, 14N
(light medium). At various times after transfer, the samples of the cells were collected
and the DNA was isolated.
The DNA molecules were fragmented during isolation.

In semiconservative mode, after one generation all the daughter molecules would have
one 15Nand one 14N strand called as hybrid molecule.
Hence all the daughter molecules would have same density ( hybrid density )- namely
midway between that of (15N15N) and (14N14N) molecules.
When DNA was extracted from bacteria and its density measured by centrifugation in
CsClas function of time after the change from heavy to light medium, the result
obtained showed that all DNA had a hybrid density after one round of replication,
indicated that semiconservative mode is correct.
The second experiment confirmed the structure of the (15N14N) DNA found after one
generation. In this experiment the hybrid DNA was denatured by heating to 1000 C and
centrifugedin CsCl.
The heated DNA yielded two bands having the densities of denatured single stranded
(15N) and (14N) DNA of hybrid density did in fact consist of one 14N and one 15N strand.
Result:
During the two generations, the DNA formed bands corresponding to its density— heavy
for parental, hybrid for the first generation, and half hybrid and half light in the second
generation.

PRIMER
Introduction
A primer is a strand segment (complementary to the template) with a free 3’-
hydroxyl group to which a nucleotide can be added. The free 3’end of the primer is
called the primer terminus. It is required during initiation process of replication.
Characteristic features
It is a part of the new strand must already be in place as all DNA polymerases can
only add nucleotides to a preexisting strand.
Most primers are oligonucleotides.
These are RNA rather than DNA.
A specialized RNA polymerase called primase forms a short RNA primer
complementary to the unwound template strand
TEMPLATE:
Introduction
All DNA polymerases require a template for DNA replication. It is required during
initiation process of replication.
Characteristic features
It is complementary to newly synthesized strand in replication.
The polymerization reaction is guided by a template DNA strand according to the
base-pairing rules.
As predicted by Watson and Crick: where a guanine is present in the template, a
cytosine deoxynucleotide is added to the new strand, and where a adenine is present
thymine is added and vice versa.
The two DNA strands are antiparallel, thus the strand serving as the template is read
from its 3’end toward its 5’end.

FIDELITY OF REPLICATION
Fidelity of Polymerases:
Fidelity exhibit varying degrees of fidelity, ranging from one misincorporation per 5000
to one per 107 nucleotides polymerized.
Those that incorporate the proper templated nucleotide at high efficiency are termed
high-fidelity enzymes, and those that frequently misinsert a nucleotide are termed low-
fidelity. Several polymerases contain a 3′-5′ exonuclease subdomain (ie, a proofreading
subunit) which increases the fidelity of the enzyme by approximately 10- to 100-fold.
The fidelity of polymerases is determined by one of several procedures. Fidelity of DNA
synthesis was initially measured by utilizing polynucleotide templates consisting of only
one or two types of nucleotides, such as an alternating poly d(A-T) template, and
measuring the extent of misincorporation of radioactive cytosine or guanine
nucleotides.
Greater sensitivity has been obtained with biological reversion assays, in which
misincorporation by DNA polymerase results in the converting an amber mutation (ie,
stop codon) in a plasmid into one that encodes an active, full-length protein.
The forward mutational assays developed more recently offer the additional advantage
of determining the mutational spectrum, that is, the types of misincorporated
nucleotides catalyzed by the polymerase.
LacZ has been most extensively utilized in these forward mutational assays as a
reporter gene for studies on the mutational spectrum of DNA polymerases.
Upon transformation of the copied plasmid (which encodes the LacZ gene) into E. coli
and plating the transfected bacteria in the presence of X-gal (which is converted to a
blue staining metabolite by the protein encoded by the LacZ gene, b-galactosidase), the
fidelity is determined simply by counting the number of blue and white colonies
resulting from functional (or nonmutated) or nonfunctional (or mutated) LacZ gene,
respectively. Sequencing the LacZ gene mutants determines the mutational spectrum.

The fidelity of incorporation is also determined kinetically by comparing the ratio k
cat/Km of the incorrect nucleotide to that of the correct nucleotide, this ratio directly
reflects the efficiency of nucleotide incorporation.
As a second step, the same assay measures the fidelity of extension by using primers
that terminate in a noncomplementary nucleotide and measuring the incorporation of
complementary nucleotides onto the end of this primer.
Processivity:
Processivity refers to the number of nucleotides incorporated per binding event of the
polymerase with the template-primer complex. The processivity values of different
polymerases range from one nucleotide to about ten thousand. The processivities of
several polymerases involved in genomic replication are enhanced upon binding to a
second protein, termed the processivity factor.
For example, to fulfill their roles efficiently during DNA replication in eukaryotes, DNA
polymerases d and associate with a homotrimer that has 36-kDa subunits of
proliferating cellular nuclear antigen (PCNA) which form a “sliding clamp”. Phage T4
gene 45 protein and E. coli beta similarly augment the processivity of T4 DNA pol and
pol III, respectively, by acting as “sliding clamps” bound to the polymerase, thus
preventing its dissociation from DNA.
PROOFREADING:
One mechanism intrinsic to virtually all DNA polymerases is a separate 3’→5’
exonuclease activity that double-checks each nucleotide after it is added. This nuclease
activity permits the enzyme to remove a newly added nucleotide and is highly specific
for mismatched base pairs .
If the polymerase has added the wrong nucleotide, translocation of the enzyme to the
position where the next nucleotide is to be added is inhibited. This kinetic pause
provides the opportunity for a correction. The 3’→5’ exonuclease activity removes the
mispaired nucleotide, and the polymerase begins again.
This activity, known as proofreading, is not simply the reverse of the polymerization
reaction because pyrophosphate is not involved.
The polymerizing and proofreading activities of a DNA polymerase can be measured
separately.
Proof reading improves the inherent accuracy of the polymerization reaction 10² to 10³
fold.

In the monomeric DNA polymerase I, the polymerizing and proofreading activities have
separate active sites within the same polypeptide.
When base selection and proofreading are combined, DNA polymerase leaves behind
one net error for every 106 to 108 bases added. Yet the measured accuracy of
replication in E. coli is higher still.
The additional accuracy is provided by a separate enzyme system that repairs the
mismatched base pairs remaining fter replication
NEAREST NEIGHBOR FREQUENCY ANALYSIS

Introduction:
Techniques of sequence analysis were available two decades before.However, it did not
measure the fidelity of copying the correct sequence.This frequency can be determined
by nearest neighbour analysis.
Principle:
The technique of frequency analysis of nearest neighbour base sequences in DNA (Josse, Kaiser
& Kornberg, I96t) allows the characterization and description of a DNA in terms of the average
frequency of occurrence of its sixteen possible doublet sequences
This procedure is based on the incorporation of [α-32p] dNTPs, followed by enzymatic
hydrolysis of the product to 3’- deoxynucleotides.
Method:
This method is specially used for DNA and hence requires the synthesis of DNA by DNA
polymerases and 4 5’-deoxyribonucleoside triphosphates, one of which is labeled with
32p at α phosphorous.
Steps:
1. DNA is synthesized in the presence of α-radiolabelled (32p) precursor(Eg: CT-*P-P-P)
2. Radiolabelled phosphorous is incorporated into the phosphodiester bond on the 5’ side
of this and all other cytosines incorporated.
3. The synthesized DNA is digested.
4. Phosphodiester bonds are broken; mononucleotide with phosphates attached attached
to their 3’-OH areabtained.
5. The radiolabelled phosphorous is this passed to its nearest neighbour
6. This would result in all 32p labeled (A, T, G and C).
7. All four radiolabelled nucleotides are repeated.
8. Radioactivity in each is determined.
9. Thus, cytosine nearest neighbour frequency is determined.
Detailed explanation:
Strategy of an experiment to trace the origin of the phosphate between the two
exons in a spliced transcript.

(a) Nearest neighbor analysis. This technique is not the one Sharp and colleagues used,
but it embodies the same principle.
First, label RNA by synthesizing it in the presence of an α-32P-labeled nucleotide, CTP
in this case. The labeled phosphate (red) is incorporated between the C and its nearest
neighbor on the 5′-side, a U in this case. Next, hydrolyze the labeled RNA with alkali,
which cleaves on the 3′-side of every base, yielding mononucleotides. Notice that this
transfers the labeled phosphate to the nearest neighbor on the 5′-side, so the uracil
nucleotide is now labeled instead of the cytosine nucleotide. Finally, separate all four
nucleotides and determine the radioactivity in each. This tells how frequently each
nucleotide is C’s nearest neighbor on the 5′-side.
(b) Identifying the origin of the phosphate between spliced exons.
Step 1: Sharp and his colleagues labeled the splicing precursor with [α-32P] CTP,
which labels the phosphate (red) between the intron and the second exon. Step 2:
Our hypothesis of splicing places the labeled phosphate in the bond between the two
spliced exons. The alternative scheme would involve the phosphate originally
attached to the end of the first exon (blue in step 1), which would not be labeled
because it entered the RNA on a GTP (the first G in the intron). Step 3: Next, Sharp
and coworkers cleaved the spliced RNA with RNase A, which cuts after the pyrimidine
C, yielding the oligonucleotide GpGpGpCp, in which the phosphate between the last
G and the C is labeled (red). Finally, they cleaved this oligonucleotide with alkali,
cutting it into individual nucleotides. They analyzed these nucleotides for
radioactivity and found that GMP was labeled. This indicated that the labeled
phosphate at the end of the intron really did form the bridge between spliced exons in
the final RNA.

EUKARYOTIC DNA POYMERASES AND MECHANISM OF REPLICATION
Introduction
• A replication fork (Growing point) is the point at which strands of parental duplex
DNA are separated so that replication can proceed.
• A complex of proteins including DNA polymerase is found at the fork.
• When the circular DNA chromosomeof E. coli is copied, replication begins at a single
point, theorigin. Synthesis occurs at the replication fork, the place atwhich the DNA
helix is unwound and individual strands are replicated.
• Two replication forks move outward from the origin untilthey have copied the whole
replicon, that portion of the genome that contains an origin and is replicated as a
unit. When the replicationforks move around the circle, a structure shaped like
theGreek 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.
• In both bacteria and mammals replication forks originate at a structure called a
replication bubble,a local region where the two strands of the parental DNA helix have
been separated from eachother to serve as templates for DNA synthesis
Events occring

• During replication the DNA double helix must be unwound togenerate separate single
strands. Helicaseswhich binds to atrich region of DNA called replication origins,
are responsible for DNA unwinding. These enzymes useenergy from ATP to unwind
short stretches of helix just ahead of thereplication fork. Once the strands have
separated, they are kept single through specific binding with single-stranded DNA
bindingproteins (SSBs)
• Rapid unwinding can lead to tension and formation of supercoils or supertwists in
the helix. The tension generated by unwinding is relieved, and the unwinding process
is promoted by enzymes known as topoisomerases.
• DNA gyrase is an E. coli topoisomerase that removes the supertwists produced
during replication.
• DNA is probably replicated continuously by DNA polymerase III when the leading
strand is copied. Lagging strand replication is discontinuous, and the fragments are
synthesized in the 5′ to 3′ direction just as in leading strand synthesis.
• First, a special RNA polymerase called a primase synthesizes a short RNA primer,
usually around 10 nucleotides long, complementary to the DNA. It appears that the
primase requires the assistance of several other proteins, and the complex of the
primase with its accessory proteins is called the primosome.
• DNA polymerase III holoenzyme then synthesizes complementary DNA beginning at
the 3′ end of the RNA primer.

• In order for DNA polymerases to move along and copy a duplex DNA, helicase must
sequentially unwind the duplexand topoisomerase must remove the supercoils that
form.
• A major complication in the operation of a DNA replicationfork arises from two
properties: the two strands of theparental DNA duplex are antiparallel, and DNA
polymerases (like RNA polymerases) can add nucleotides to thegrowing new strands
only in the 5’→3’ direction.
• Synthesisof one daughter strand, called the leading strand, can
proceedcontinuously from a single RNA primer in the 5’→3’direction, the same
direction as movement of the replicationfork. The problem comes in synthesis of
theother daughter strand, called the lagging strand.
• A cell accomplishes lagging strand synthesis by synthesizing a new primer every few
hundred bases or so on the second parental strand, as more of the strand is exposed
by unwinding. Each of these primers, base-paired to their template strand, is
elongated in the 5’→3’ direction, forming discontinuous segments called Okazaki
fragments.
Termination of replication:

• DNA replication stops when the polymerase complex reaches a termination site on
the DNA in E. coli. The Tus protein binds to these Tersites and halts replication. In
many procaryotes, replication stops randomly when the forks meet.
• Eventually, the two replication forks of the circular E. coli chromosome meet at a
terminus region containing multiple copies of a 20 bp sequence called Ter (for
terminus). The Ter sequences are arranged on the chromosome to create a sort of
trap that a replication fork can enter but cannot leave. The Ter sequences function as
binding sites for a protein called Tus (terminus utilization substance). The Tus-Ter
complex can arrest a replication fork from only one direction.
• Only one Tus-Ter complex functions per replication cycle—the complex first
encountered by either replication fork. Given that opposing replication forksgenerally
halt when they collide, Ter sequences do notseem essential, but they may prevent
overreplication byone replication fork in the event that the other is delayedor halted
by an encounter with DNA damage orsome other obstacle.
• When either replication fork encounters a functional Tus-Ter complex, it halts; the
other fork halts when it meets the first (arrested) fork.
• The final few hundred base pairs of DNA between these large protein complexes are
then replicated (by an as yet unknown mechanism), completing two topologically
interlinked (catenated) circular chromosomes. DNA circles linked in this way are
known as catenanes. Separation of the catenated circles in E. coli requires
topoisomerase IV (a type II topoisomerase). The separated chromosomes then
segregate into daughter cells at cell division. The terminal phase of replication of
other circular chromosomes, including many of the DNA viruses that infect
eukaryotic cells, is similar.
TELOMERE SYNTHESIS – TELOMEREASES
Telomeres (Greek telos, “end”) are sequences at the ends of eukaryotic chromosomes
that help stabilize the chromosome.
Features
Telomeres, the structures at the ends of linear eukaryotic chromosomes generally
consist of many tandem copies of a short oligonucleotide sequence. There may be
100-1000 repeats, depending on the organism.

One unusual property of the telomeric sequence is the extension of the G-T-rich
strand, usually for 14-16 bases as a single strand. The G-tail is probably generated
because there is a specific limited degradation of the C-A-rich strand.
The single-stranded G-rich tail of the telomere can form "quartets" of G residues.
Each quartet contains 4 guanines that hydrogen bond with one another to form a
planar structure.
Loop of DNA is formed at the telomere. The absence of any free end may be the
crucial feature that stabilizes the end of the chromosome. The average length of the
loop in animal cells is 5-10 kb.
Role
The need for a specialized region at the ends of eukaryotic chromosomes is apparent
as all known DNA polymerases elongate DNA chains at the 3’ end, and all require an
primer. As the growing fork approaches the end of a linear chromosome, synthesis of
the leading strand continues to the end of the DNA template strand, completing one
daughter DNA double helix.
Telomerase:
Telomeres, the structures at the ends of linear eukaryotic chromosomes generally
consist of many tandem copies of a short oligonucleotide sequence.
This sequence usually has the form TxGy in one Strand and CyAx in the
complementary strand, where x and y are typically in the range of 1 to 4.
Telomeres vary in length from a few dozen base pairs in some ciliated protozoan’s to
tens of thousands of base pairs in mammals.
The TG strand is longer than its complement, leaving a region of single-stranded DNA of
up to a few hundred nucleotides at the 3’ end. The ends of a linear chromosome are not
readily replicated by ate and primer, and beyond the end of a linear DNA molecule no
template is available for the pairing of an RNA primer.

Without a special mechanism for replicating the ends, chromosomes would be
shortened somewhat in each cell generation. The enzyme telomerase solves this
problem by adding telomeres to chromosome ends.
Telomerase
The problem of telomere shortening is solved by an enzyme that adds telomeric
sequences to the ends of each chromosome. The enzyme is a protein and RNA
complex called telomere terminal transferase, or telomerase.
Thus telomerase is a specialized form of a reverse transcriptase that carries its own
internal RNA template to direct DNA synthesis.
Telomerase, by reverse transcription of its associated RNA, elongates the 3’ end of the
single-stranded DNA at the end of the G-rich strand.
DNA loopOR TELEMORE (‘t’) loop
For years, molecular biologists pondered this question and, as telomere-binding
proteins were discovered, they theorized that these proteins bind to the ends of
chromosomes and in that way identify the ends.

One problem with this hypothesis is that the mammalian telomere binding proteins,
such as the TTAGGG repeat-binding factors TRF1 and TRF2 bound duplex DNA in
the telomeres specifically. No one could find a mammalian protein that was specific for
the very end of the telomere.
Then, in 1999, Jack Griffith and Titia de Lange and their colleagues discovered that
telomeres are not linear, as had been assumed, but form a DNA loop they called a t
loop (for telomere loop). These loops are unique in the chromosome and therefore quite
readily set the ends of chromosomes apart from breaks that occur in the middle and
would yield linear ends to the chromosome fragments.
Griffith, de Lange and colleagues started by making a model mammalian telomeric DNA
with about 2 kb of repeating TTAGGG sequences, and a 150–200-nt single stranded 3′-
overhang at the end. They added one of the telomere-binding proteins,
TRF2, then subjected the complex to electron microscopy.
It shows that a loop really did form, with a ball of TRF2 protein right at the loop– tail
junction. Such structures appeared about 20% of the time. By contrast, when these
workers cut off the singlestranded 3′-overhang, or left out TRF2, they found a drastic
reduction in loop formation.
One way for a telomere to form such a loop would be for the single-stranded 3′-
overhang to invade the double stranded telomeric DNA upstream.
If this hypothesis is correct, one should be able to stabilize the loop with psoralen and
UV radiation, which cross-link thymines on opposite strands of a double stranded DNA.
Because the invading strand base-pairs with one of the strands in the invaded DNA this
creates double-stranded DNA that is subject to cross-linking and therefore stabilization.
The results of an experiment in which Griffith, de Lange, and coworkers cross-linked
the model DNA with psoralen and UV, then deproteinized the complex, then subjected it
to electron microscopy. The loop is still clearly visible, even in the absence of TRF2,
showing that the DNA itself has been crosslinked, stabilizing the t loop. Next, these
workers purified natural telomeres from several human cell lines and from mouse cells
and subjected them to psoralen–UV treatment and electron microscopy.
They obtained the same result as showing that t loops appear to form in vivo.
Furthermore, the sizes of these putative t loops correlated well with the known lengths
of the telomeres in the human or mouse cells, reinforcing the hypothesis that these
loops really do represent telomeres.

To test further the notion that the loops they observed contain telomeric DNA, Griffith,
de Lange and colleagues added TRF1, which is known to bind very specifically to
double-stranded telomeric DNA, to their looped DNA. They observed loops coated with
TRF1.
If the strand invasion hypothesis is valid, the single-stranded DNA displaced by the
invading DNA (the displacement loop, or D loop) should be able to bind E. coli single-
strand-binding protein if the displaced DNA is long enough.
The above diagram demonstrates that SSB is indeed visible, right at the tail–loop
junction. That is just where the hypothesis predicts we should find the displaced DNA.
TRF1 and TRF2 appear to help telomeric DNA in mammalian cells form a t loop, in
which the single-stranded 3′-end of the telomere invades the double-stranded telomeric
DNA upstream.
TRF1 may help bend the DNA into shape for strand invasion, and TRF2 binds at the
point of strand invasion and may stabilize the displacement loop. The t loop structure
protects the end of the chromosome from inappropriate repair in which it would be
attached to another chromosome end
REPLICATION OF VIRAL DNA
Replication of various human adenoviruses entry takes place via interactions of the
fiber knob with specific receptors on the surface of a susceptible cell followed by
internalization via interactions between the penton base and cellular integrins. After
uncoating, the virus core is delivered to the nucleus, which is the site of virus
transcription, DNA replication, and assembly.
Virus infection mediates the shutdown of host DNA synthesis and later RNA and
protein synthesis. Transcription of the adenovirus genome by host RNA polymerase II

involves both DNA strands of the genome and initiates (in HAdV-2) from five early (E1A,
E1B, E2, E3, and E4), two intermediate, and the major late (L) promoter.
All primary transcripts are capped and polyadenylated, with complex splicing patterns
producing families of mRNAs. In primate adenoviruses, one or two VA RNA genes are
usually present upstream from the main pTP coding region. These are transcribed by
cellular RNA polymerase III and facilitate translation of late mRNAs and blocking of the
cellular interferon response.
Corresponding VA RNA genes have not been identified in nonprimate adenoviruses,
although a nonhomologous VA RNA gene has been mapped in some aviadenoviruses
near the right end of the genome. More generally, the replication of aviadenoviruses has
been shown to involve significantly different pathways from those characterized in
human adenoviruses.
This is not unexpected, given the considerable differences in gene layout between
nonconserved regions of the genome.
About 40 different polypeptides (the largest number being in fowl adenoviruses and the
smallest in siadenoviruses) are produced. Almost a third of these compose the virion,
including a virus-encoded cysteine protease.
1. Adsorption of virions to the cell surface
2. Entry by endocytosis
3. Transport to the cell nucleus (route and mechanism not yet known);
4. Uncoating;
5. Transcription to produce early region mRNAs;
6. Translation to produce early proteins (T antigens);
7. Viral DNA replication;
8. Transcription to produce late region mRNAs;
9. Translation to produce late proteins (capsid proteins);
10. Assembly of progeny virions in the nucleus;
11. Entry of virions into cytoplasmic vesicles (mechanism unknown);
12. Release of virions from the cell by fusion of membrane vesicles with the plasma
membrane;
Released virion. Virions are most likely also released from cells at cell death when
virions have an opportunity to leak out of the nucleus.

In nonpermissive cells, the first six steps occur normally, but viral DNA replication
cannot occur and subsequent events do not take place.
M13 BACTERIOPHAGES REPLICATION
In DNA replication, the DNA polymerase cannot initiate the synthesis of a new DNA
strand and must rely on a priming device.
In general, an RNA primer is synthesized at or near a replication origin to start
synthesis of the leading strand. However, a DNA primer terminus can be generated by
a nuclease-generated nick at a specific place in some circular duplex DNA, and
replication will then proceed unidirectionally, as shown in Figure. This mode of
replication is called rolling circle replication and is found for replication of the replicative
form (RF) form of bacteriophage singlestranded genomes of Gram-negative bacteria and
of the multicopy plasmids of Gram-positive bacteria.
Rolling circle replication is also observed in the late stage of the replication of the
lambda phage genome and in the process of the conjugative transfer of bacterial
plasmids.
DNA synthesis initiates using the free 3′-OH end at the nick as a primer, and a
replication fork proceeds around the template. In the process, the newly synthesized
strand displaces the old strand from the template. In the case of replication of the RF
form of single-stranded phage genomes and of plasmids of Gram-positive bacteria, the
displaced old strand is cleaved off after one round of replication and is converted into
the circular, double-stranded form. In contrast, in phage lambda replication, the
replication fork precedes a number of revolutions around the template without cleavage
of the displaced strand, and the displaced strand becomes double-stranded as it is

peeled off. The linear concatemer thus created is cleaved into one unit length and
packaged into the phage particles. In the conjugation process of plasmids, the displaced
strand is transferred into the new cell.
ROLLING CIRCLE MODEL
Phage ØX174 consists of a single-stranded circular DNA, known as the plus (+) strand.
A complementary strand, called the minus (-) strand, is synthesized. This action
generates the duplex circle shown at the top of the figure, which is then replicated by a
rolling circle mechanism.
The duplex circle is converted to a covalently closed form, which becomes supercoiled. A
protein coded by the phage genome, the A protein, nicks the (+) strand of the duplex
DNA at a specific site that defines the origin for replication.
After nicking the origin, the A protein remains connected to the 5' end that it generates,
while the 3' end is extended by DNA polymerase.
The structure of the DNA plays an important role in this reaction, for the DNA can be
nicked only when it is negatively supercoiled.
The A protein is able to bind to a single-stranded decamer fragment of DNA that
surrounds the site of the nick. This suggests that the supercoiling is needed to assist
the formation of a single-stranded region that provides the A protein with its binding
site. (An enzymatic activity in which a protein cleaves duplex DNA and binds to a
released 5' end is sometimes called a relaxase. The nick generates a 3'-OH end and a 5'-

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