Nomenclature

Nucleoside Nucleotide
Base +deoxyribose +phosphate
Purines
adenine adenosine
guanine guanosine
hypoxanthine inosine
Pyrimidines
thymine thymidine
cytosine cytidine

+ribose
uracil uridine 15
 “Why is thymine found only in DNA, uracil only in RNA?”

Deamination of cytosine in DNA is very common in the cell,

but can lead to mutations after DNA replication.

deamination
C:G U:G
DNA DNA
replication replication

C:G U:T & C:G

There is a repair system that looks for uracil attached to

deoxyribose sugars and excises the base then replaces the uracil
with a cytosine.

If uracil was a normal base in DNA, the cell might not distinguish the uracil
normally present and uracil from the code. the U:G bps
17
18
6Å
3.4Å

34Å

Helix vs Spiral
DNA is:
1) double stranded
2) 2 strands are anti-parallel
20
 B DNA

Z DNA
 3 forms of DNA

– –Z B
A form:form
form: formed
theamost
leftinhanded
likely
the laboratory
biological when
conformation,
helix; some water
evidence
concentration
right
that handed
this helix
is foundisindecreased (more
living cells, compact double
but implication of
helix);
this formright handed helix
unknown
24
 DNA structure:
The genetic information of all living organisms except the RNA viruses, is
therefore apparently stored in DNA.

Nucleic acids were first called “nuclein” since they were isolated from cell
nuclei by F. Miescher (1869).

By 1930, Caspersson had shown that DNA consists of extremely large

molecules (much larger than proteins). But monotony of its structure in all
organisms appeared to exclude any possible genetic role for which lot of
variation is expected like proteins.

Continuing analysis carried out by Chargaff demonstrated that bases present

in different species are in different amounts (1951).
Species % % % % A+G/ T+C A+T/ G+C
Adenine Guanine Cytosine Thymine

Bacteriophage l 26.0 23.8 24.3 25.8 0.99 1.08

Bacteriophage T2 32.6 18.1 16.6 32.6 1.03 1.88
Herpes virus 13.8 37.7 35.6 12.8 1.06 0.36
E. coli 26.0 24.9 25.2 23.9 1.04 1.00
Diplococcus pneumoniae 29.8 20.5 18.0 31.6 1.02 1.59
Neurospora 23.0 27.1 26.6 24.3 1.00 0.86
Aspergillus 25.0 25.1 25.0 24.9 1.00 1.00
Yeast 31.7 18.3 17.4 32.6 1.00 1.80
Maize 25.6 24.5 24.6 25.3 1.00 1.04
Tobacco 29.3 23.5 16.5 30.7 1.12 1.50
Drosophila 30.7 19.6 20.2 29.4 1.01 1.51
Man Liver 30.3 19.5 19.9 30.3 0.99 1.53
Thymus 29.8 20.2 18.2 31.8 1.02 1.60
Sperm 30.5 19.9 20.6 28.9 1.02 1.47
 % Adenine = % Thymine
% Guanine = % Cytosine

This observation led to the idea that the sequences of

bases in DNA might be the form in which genetic
information is coded that is how so much variation in
living organisms could be accounted for because the
amounts of these bases could be quite paralled in some
highly unrelated organisms like, λ phage, E. coli,
Aspergillus, and maize

and although quite significantly different between

related organism like λ phage and T 2, Maize and
Tobacco.
 The polynucleotide

Nucleic acids are the macromolecules composed of

repeating sub units called nucleotides. Each nucleotide is
composed of:

• A phosphate group
• A five carbon cycle sugar (pentose) and
• A heterocyclic nitrogen containing base.
Nitrogenous bases:

fall into two types

Purines and Pyrimidines

A,G,C : in both DNA & RNA

T : in DNA only
U : in RNA only

Purines: have fused five and six member U rings.

Pyrimidines: have six member C rings.
The purines and pyrimidines can be seen to contain several unsaturated double bonds.
Molecules containing such bonds are potentially able to exist in different chemical
forms for their hydrogen bonds have a certain freedom. In such a molecule, a hydrogen
atom can, for example, more away from an amino (-NH2) group, leaving an imino (-NH)
group and a negative charge, that is absorbed by the conjugated ring system of the
molecule. Such chemical fluctuations are called tautomeric shifts and the different
molecules are called tautomers.

The tautomeric forms of these bases rarely occur ie they remain chemically stable in
one form, which is an important genetic attribute.

Each nucleic acid is synthesized from only 4 types of bases:

Two purines
Two pyrimidins.

Same two purines (A and G) are present in both DNA and RNA.
The two pyrimidines in DNA are C and T while RNA contains U in place of T and thus has
C and U.
So, DNA has A,G,C, T -NH2---- -NH
Amino Imino
RNA has A,G,C,U -C=O----- COH
Keto enol
Pentose sugars:

Two types of pentose sugars are found in nucleic acids.

They distinguish DNA and RNA and give rise to general
names for 2 types of nucleic acids.

In DNA the pentose is 2-Deoxyribose, whereas in RNA, it

is Ribose. The difference lies in the absence / presence of
the hydroxyl group at position 2 of the sugar ring.
To avoid ambiguity between numbers of nitrogenous bases and sugars, positions in
pentose are give by primes (1`-5`)

Glycosicic bond:

The N-base is linked to position 1` on pentose ring by glycosidic bond from N1 of

pyrimidines or N4 of purines

1`-1] Glycosidic bond (Py)

1`-9] Glycosidic bond (Pu)
A base linked to a sugar is called a nucleoside and when a
phosphate group is added, the base sugar-phosphate is called
nucleotide.

The nucleotides provide the building blocks from which nucleic

acids are synthesized. The nucleotides are linked togegher into a
polynucleotide chain by a backbone consisting of an alternating
series of sugar and phosphate series (sugar-phosphate back
bone).

The 5` position of one pentose ring is connected to the 3`

position of next pentose ring via a phosphate group as shown
below. These bonds are called 5`-3` or 3`-5` phosphodiester
bonds. The nitrogenous bases stick out from the sugar
phosphate back bone.
A polynucleotide chain consists of a series of 5`-3`
sugar phosphate links that form a backbone from
which the bases protrude out.
 Abbreviated version of a polynucleotide chain

The terminal nucleotide at one end of the chain has a free 5`

group while the terminal nucleotide at the other end has a free 3`
group. The nucleotide sequence is conventionally, written in the
5`-3` direction.
 DNA is a double helix
(Watson & Crick model - 1953)

J.D. Watson and F.H.C. Crick

(1953) proposed that most
DNA is found in double
helix form and has very
specific properties. Their
double helix model of DNA
structure was based on two
major kinds of evidence.
1 When the composition of DNA from many different organisms was analysed
by E. Chargaff and co-workers, it was observed that :-
“Concentration of thymine was always equal to that of adenine (A=T) and
conc. of cytosine was always equal to that of guanine (G=C).” this indicated
their correlationships.

The total conc. of Purines (A+G) was always equal to that of Pyrimidines (T+C)

A+G = T+C ie A/T=G/C=1

While A+T/G+C ratio varied widely in DNA’s of different species

2 The X-ray diffraction pattern produced by isolated DNA fibers available from
the studies of M.H.F. Wilkins, R. Franklin and co-workers indicated that:
“DNA was a highly ordered, multiple stranded structure with repeating
substructures spaced every 3.4 angstroms (A°) along the axis of the molecule”
Photo 51 is the name given to an X-ray
diffraction image of DNA taken by Rosalind
Franklin in 1952 that was critical evidence in
identifying the structure of DNA. The photo
was taken by Franklin while working at King's
College London in Sir John Randall's group.

The photo, shown to James D. Watson by

Maurice Wilkins without Franklin's
knowledge, was the critical evidence that led
to the confirmation of the postulated double
helical structure of DNA, published during
1953 in a series of five articles in the journal Photo 51 : An X-ray diffraction
Nature. image of sodium salt of DNA. (B
configuration)

Franklin and Raymond Gosling's own

publication in the same issue of Nature was
the first publications of this more clarified X-
ray image of DNA.[
 [Watson & Crick (1953)]
[Nature 171:737-738]

“The instant I saw the picture my mouth fell open and my pulse
began to race." -- James D. Watson (1968), The Double Helix, page
167. New York: Atheneum, Library of Congress card number 68-
16217. Page 168 shows the X-shaped pattern of the B-form of
DNA, clearly indicating crucial details of its helical structure to
Watson and Crick.

On the basis of Chargaff’s chemical data, Wilkins and Franklin’s

X-ray diffraction data Watson & crick proposed that, DNA exists
as a double helix in which two polynucleotide chains are coiled
about one another in a spiral (regular helix).
Each polynucleotide chain consists of a sequence of nucleotides linked together by
phosphodiester bonds, between adjacent deoxyribose moieties.

In a phosphodiester linkage, the phosphate group present at the C-5 of the sugar of
one nucleotide gets attached to the C-3 of the sugar of the next nucleotide in the
chain.

The two polynucleotide strands are held in their helical configuration by hydrogen
bonding between the bases in opposite strands. The resulting base pairs being
stacked between the two chains perpendicular to the axis of the molecule like the
steps of a stair case.

The molecule is formed by two antiparallel polynucleotide strands which are spirally
coiled round each other in a right-handed helix. The two strands are held together
by hydrogen bonds. The double stranded helical molecule has alternate major (or
deep) and minor grooves.

The base pairing is specific ie

Adenine always paired with thymine by two hydrogen bonds (A=T).
Guanine is always paired with cytosine by three hydrogen bonds (G = C).
There, if the sequence of bases in one strand of a DNA double helix is known,
the sequence of base in the other strand is also known because of specific base
pairing. The two strands of a DNA double helix are called complementary (not
identical). This property makes DNA uniquely suited to store and transmit
genetic information.

The base pairs in DNA are stacked 3.4 A° apart with 10 base pairs per turn 360
°. So complete turn of the helix is made every 34 A ° with a diameter of about
20 A °.

The sugar-phosphate backbones of the tow complementary strands are

antiparallel i.e., they have opposite chemical polarity. As one mores
unidirectionally along a DNA double helix (5`-3`) the other complementary
strands in the opposite (3`-5`) direction.

So that bases from both strands do not protrude out in the same direction,
rather face each with for H-bonding .
 Structure of DNA (Watson and Crick model)
(A) DNA double helix. (B) Detailed structure of the two strands. (C) C-5 and C-3 ends and
antiparallel nature of strands (diagrammatic)
The high degree of stability of DNA molecule (double helix) results from the
large number of H-bonds between base pairs (even though weak) and
hydrophobic bonding ( stacking forces) between stacked base pairs in aqueous
protoplasm of living cells.

Thus each base pair is rotated 36° around the axis of the helix, relative to the
next base pair. So 10 base pairs make a complete turn of 360 °. The twisting of
the two strands around each other forms the double helix which has a
narrow grove (~12 A ° across) also called minor groove and a wide or major
groove (~22 A ° across), as can be seen from the model.

The double helix is right handed ie, the turns run clock-wise looking along the
helical axis. These features represent the accepted model for what is known
as the B-form of DNA.
 Conformational flexibility of DNA molecules

The structure of DNA generally has been considered to be one of the certainties of
molecular biology. But recently it has become clear that some of the parameters of the
classic B-form helix need to be adjusted and that DNA even may be able to exist in other
forms of double helical structures.

The vast majority of the DNA molecules present the aqueous protoplasms of living cells
certainly exist in the Watson-Crick double helix form (B form).

The B form is conformation that DNA takes under physiological conditions of aqueous
solution containing low concentration of salts. However DNA is not a static invariant
molecule. Rather DNA molecules exhibit a considerable amount of conformational
flexibity.

The structure of DNA molecule changes as a function of their environment. The exact
conformation of a given DNA molecule or segment of DNA molecule will depend on
the nature of the molecules with which it is interacting.
Because of this variation, there appear to be families of structures, each of a
characteristic type, but showing difference in governing parameters.
N: no of nucleotides per turn
H: distance between adjacent repeating units.
The variation is achieved by changes in the rotation groups about bonds with
rotational freedom.
 Structural Families in which DNA can exist

Helix Handedness Base Vertical Rotation Helical Conditions for existence

type of Helix pairing rise (deg) per bp diameter
per turn per bp A0
A Right 11 2.56A0 +32.70 23 Relative humidity 75%
Very high concentration of Na, K or
Cs ions
Very close to DNA-RNA hybrids or
RNA-DNA d/s regions
B Right 10-10.6 3.36A0 +36.00 19-20 Relative humidity 92% (very high)
Low ionic strength present in
majority of living cells.
C Right 9*1/3 3.32A0 +38.60 19 Relative humidity 66% (low)
Li ions
Z Left 12 3.71A0 -30.00 18 Zig Zag, in high salt conc.
In DNA or Poly d (GC CG)n & poly
d(AT TA)n types present in minority
in living cells.

In the D-form of DNA there are 8 bp per turn of helix and axial rise is 3.03 A°
From left to right, the structures of A, B and Z DNA
DNA forms :

Living cells full of H2O (high Relative humidity) and ionic strength not very high B-DNA

Relative humidity rather low, salt concentration is very high

(saturated with Na, K, Cs ions) A-DNA

Lower Relative humidity, Li ions present C-DNA

Very high concentration of salts (stress), poly d (GC CG)n and poly d (AT TA)n Z-DNA
Z-DNA has ------GCGCGCGCGC----or -----ATATATAT----
Zig-Zag path ------CGCGCGCGCG----- -----TATATATA----
of chain K
(Salivary gland chromosomes Antibodies against Z-form can distinguish it
of Drosophila) from B-form.
References

Chargaff, E.K (1951). Fed. Proc. 10:654-659

Davidson, J.N. (1972). The Biochemistry of Nucleic Acids 7th ed.

Chapman Hall

Wilkins, M.H.F., Strokes, A.R. and Wilson, H.R. (1953). Nature

171:738-740.
 DNA Replication in prokaryotes

❑Introduction to Replication
❑Replication process in Prokaryotes
❑Proposed models of replication
❑ Semi-conservative method by Meselson
& Sthal
Introduction
• DNA replication, the basis for biological
inheritance, is a fundamental process occurring in
all living organisms to copy their DNA.
• In the process of "replication" each strand of the
original double-stranded DNA molecule serves as
template for the reproduction of the
complementary strand.
• Two identical DNA molecules have been
produced from a single double-stranded DNA
molecule.
• DNA has to be copied before a cell divides
• DNA is copied during the S or synthesis phase
of interphase
• The unit of DNA replication is referred to as a
replicon

DNA replication takes place in S phase

 Replicon
• Replicon is the segment of DNA that is capable of DNA
replication
• Each replicon has an origin and terminus
• Bacterial and viral chromosomes usually contain a single
replicon per chromosome
• In Eukaryotes each chromosome is made up of several
replicons ex: 3500 replicons in 4 chromosomes of
Drosophila
 Origin of Replication
• Replication begins at one site called origin (ori) and proceeds
in both directions around the chromosome. It is a particular
sequence in a genome at which replication is initiated.
• Origins are A T rich which is important for easy un winding
of the two strands.
• Origin of replication in E. coli is ori C locus its 245 bp long.

Terminus
• The site at which replication stops.
• E. coli has two termini one on each strand.
• Termination requires tus gene product , which recognize and
binds to ter sequence and stops replication.
BIDIRECTIONAL REPLICATION
• Synthesis of DNA proceeds bidirectionally
around the bacterial chromosome.
• The chromosome of E. coli phage T7
replicates in a linear form, and DNA
replication begins at one end.
• Replication produces so called eye
structure in the t7 chromosome
• Two replication forks are formed and
progress in the both the directions away
from the origin.
• Replication fork will reach one end of the
chromosome much sooner than the other end
& give rise to Y shaped chromosome.
• Melting produces two Y shaped forks at origin; one
located at each end of origin.
• These forks become replication forks when replication
begins
• Replisome is the smallest functional unit in the
factories and are responsible for copying one segment
of DNA
• To begin DNA replication, unwinding enzymes called
DNA helicases cause the two parent DNA strands to
unwind and separate from one another at the origin of
replication to form two "Y"-shaped replication forks.
• The double helix is unwound by the enzymes helicase , and
DNA gyrase
• SSBP (single stranded binding protein) helps keep strands
separated
• DNA polymerase III (pol III) is responsible for most of DNA
synthesis
– Adds nucleotides to the 3’ end of the daughter strand of
DNA; DNA synthesis is from 5' to 3‘
– Requires RNA primers as a guide for synthesis
• RNA primers are made by the enzyme primase
• DNA polymerase I: involved in proof reading and DNA repair
• DNA ligase: involved in connected ends of replicated DNA
together
 Replication process in Prokaryotes
• DNA replication includes:
• Initiation – replication begins at an origin of
replication
• Elongation – new strands of DNA are
synthesized by DNA polymerase
• Termination – replication is terminated
differently in prokaryotes and eukaryotes
A] INITIATION
The main events involved in replication initiation
are:
I. DnaA recognizes oriC seq. and
opens duplex at specific sites.
Denaturation of DNA at oriC
requires a histone-like HU
protein and ATP
II. DnaB helicase (hexamers) binds
to unwound DNA region.
Binding of DnaB to individual
strands also requires DnaC.
III. DNA gyrase unwinds DNA
IV. SSBs bind to single stranded
DNA
v. DnaG primase synthesizes
short RNA primers.
INITIATION in detail
• 2-4molecules of DnaA bind oriC results in folding of
DNA around the aggregate & melting starts
• An aggregate having 6 molecules each of DnaB and
DnaC binds to each of the three separate single-
standard regions produced by DnaA
• Agregate eventually displaces DnaA and DnaC loads
DnaB
• DnaB funtions as Helicase and begin unwinding and
Gyrase provides a swivel
• SSBP bind to single strand regions and stabilise them
• One DnaB hexamer binds to each of the two folks
produced by unwinding at the origin
 INITIATION in detail
• Once replication fork is generated primosome
assembles the origin, and initiates primer
synthesis(priming)
• Priming occurs only once and at origin for the
replication of leading strand
• Priming occurs repeatedly at intervals of 1000 to 2000
bases in case of lagging strand
• The primosome moves along the single standard region
• When the primosome reaches a site at which a priming
can occur, it synthesizes an RNA primer and the primer
sponsors synthesis of a new okazaki fragments
➢Melting of DNA by DnaA
➢Release of DnaB at the forks by DnaC
➢Helicase action of DnaB
➢Swivel action of DNA Gyrase
➢Activation of primase DnaG by DnaB
➢Activation of DNA polymerase to start
replication
B] ELONGATION
Requires at least 7 proteins
a) SSBs bind to ssDNA
b) DnaB helicase unwind DNA, primosome
c) Primase RNA primer synthesis
d) DNA pol. III new strand elongation
e) DNA pol. I remove primers & fills gaps
f) DNA ligase seals nicks
g) DNA gyrase supercoiling
 Subunit Function
α DNA polymerase
ε 3’ – 5’ Exonuclease CATALYTIC
CORE
θ stimulates 3’ – 5’ exonuclease
τ dimerises core and activates helicase
γ binds ATP
δ&ψ Unknown CLAMP

χ
LOADER
Removal of primase
β Sliding clamp
A “clamp loader:”
complex is required to get
the clamp onto the DNA
ELONGATION in detail

• The entire DNA-synthesizing complex at each

replication fork, which also includes
topoisomerase, helicase, and primase, is
referred to as a replisome
• Replication activity is due to the DNA
polymerase III of Replisome.
• E.coli has 10 molecules of DNA polymerase
enzymes
 ELONGATION in detail

• A single holo enzyme molecule functions at one replication

fork.
• Each holo enzyme molecule has 2 catalytic cores, one
catalyses the replication of leading strand and other catalyses
lagging strand.
• In case of leading strand the catalytic core extends the primer
one nucleotide at a time.
• DnaB progressively unwinds the duplex and the replication
fork moves along
• Replication of lagging strand will be after some time.
• When DnaB associated with the advancing fork reaches a site
suitable for priming,it activates DnaG to synthesize a primer
in the normal 5’-3’ direction i.e, towards oriC
• When the primer becomes10-14bases the other
core begins to elongate in 5’-3’ direction
• The lagging strand is in effect , pulled up by the
replisome in the process of replication
• When the replisome reaches 5’-end of primer of
previous okazaki fagment it stops replication and
dissociates from the lagging strand
• Mean while DnaB continues to move forward
with the replication fork
• When it reaches the appropriate site, it again
induces primer synthesis by DnaG and the events
described above takes place again
Simultaneous synthesis of leading and lagging strands:

• DNA is synthesized only in the 5’-3’ direction and never in the

3’-5’ direction for the complementary strand
• In the 3’-5’ template strand the DNA synthesis made in the
direction in a continuous way is called the
• On the other template short pieces of DNA ( ~1000 nucleotides
long) in the 5’- 3’ direction are made and the pieces are joined
together.
• The small fragments of DNA are called Okazaki fragments,
after their discoverer, R. Okazaki.
• The new DNA is made by this discontinuous method is called
the lagging strand
• The leading and lagging strands are synthesized simultaneously
by a single dimeric DNA polymerase III complex
Simultaneous synthesis of both DNA
strands
DNA Chain extension:
• The primer is then extended by DNA Pol III
• DNA Pol synthesizes DNA for both the
leading and lagging strands
• After DNA synthesis by DNA Pol III DNA
pol I uses its 5’-3’ exonuclease activity to
remove the primer and then fills the gaps with
a new 5’ – 3’ exonuclease activity
• Finally, DNA pieces are joined together by the
DNA ligase
DNA ligase seals the gaps between Okazaki
fragments with a phosphodiester bond
C] TERMINATION
• The sequence that stop the movement of
replication fork are identified as ‘ter’ elements
• These are 23 bp consensus sequences that provide
the binding site for the ‘tus’ gene, a 36 kD Protein
needed for the termination.
• Tus protein binds to ter elements and stops Dna B
from unwinding DNA.
• Leading strand replicated upto ter elements and
lagging strand replication stopped 50-100bp
before the ter elements.
• In the late 1950s, three different mechanisms were
proposed for the replication of DNA
➢ Conservative model
• Both parental strands stay together after DNA
Replication
➢ Semi-conservative model
• The double-stranded DNA contains one parental and
one daughter strand following replication
➢ Dispersive model
• Parental and daughter DNA are interspersed in both
strands following replication
Semi-conservative model Conservative model Dispersive model
The double-stranded DNA Both parental Parental and daughter
contains one parental and strands stay DNA are interspersed in
one daughter strand together after both strands following
following replication DNA replication replication
• Bacterial (E. coli) DNA is placed in a media containing heavy
nitrogen (N15), which binds to the DNA, making it identifiable.
• This DNA is then placed in a media with the presence of N14 and
left to replicate only once. The new bases will contain nitrogen 14
while the originals will contain N15.
• The DNA is placed in test tubes containing cesium chloride (heavy
compound) and centrifuged at 40,000 revolutions per minute.
• The cesium chloride molecules sink to the bottom of the test tubes
creating a density gradient. The DNA molecules will position at
their corresponding level of density (taking into account that N15 is
more dense than N14)
• These test tubes are observed under ultraviolet rays. DNA appears
as a fine layer in the test tubes at different heights according to their
density.
1958: Matthew Meselson & Frank Stahl’s Experiment
Semiconservative model of DNA replication
Chromosomes in Eukaryotic cells consist of:

✓DNA
✓Protein
✓some
chromosomal
RNA
DNA Replication machinery involved in
Eukaryotes
• DNA Polymerases α,δ,ε,γ.
• Helicase
• Topoisomerase
• Primase
• Ligase
• SSB Proteins
• dNTPs, Mg+2, & ATP
Protein Role
DNA helicases Unwinds the double helix
RNA primase Synthesizes RNA primers
Single-strand Keep the two strands separates
binding proteins
DNA polymerase α Add RNA primers during
initiation
DNA polymerase δ Proofreading of DNA, add
Okazaki fragments
DNA polymerase ε Synthesizes DNA, proofreading
DNA ligase Joins the ends of DNA
fragments; DNA repair
 DNA Polymerase α
• Involved in initiation
• Synthesizes an RNA primer then adds dNTPs
• A complex of four subunits 50-kD and 60-kD
are primase subunits;180-kD subunit DNA
polymerase, Synthesizes 10-12 nt RNA
primers.
• synthesizes RNA primers for the leading
strands and each lagging strand fragments.
 DNA Polymerase δ
• The principal DNA polymerase, add okazaki
fragments in lagging strand.
• Has role in proofreading.
• Consists of a 125 kD and a ~50 kD subunit.
• The 50kd subunit interacts with PCNA
(Proliferating Cell Nuclear Antigen).
 DNA Polymerase ε
• Catalyze replication of leading strand ,also contribute
in proofreading.
• DNA Polymerase ε Consist of more than one subunit,
>300 kd
• Polymerases δ and ε both contain the 3’ 5'exonuclease
activity required for proof reading.

DNA Polymerase β:
• Monomeric having 36-38 kd Involved in DNA repair
process.
DNA Polymerase γ:
• The DNA-replicating enzyme of mitochondria
 Proteins Involved in Eukaryotic
DNA Synthesis:
PCNA (Proliferating Cell Nuclear Antigen)
Provide substrate for DNA Polymerase δ, the
eukaryotic counterpart of the Sliding Clamp of E. coli
PCNA also encircles the double helix.
RPA (Replication Protein A)
ssDNA-binding protein that facilitates the unwinding of
the helix to create two replication forks ,the eukaryotic
counterpart of the SSB protein.
RFC (Replication Factor C)
The eukaryotic counterpart of the complex Clamp
Loader of E. coli that is it loads PCNA on DNA .
 Proteins Involved in Eukaryotic
DNA Synthesis:
ORC ( Origin recognition complex)
Multisubunit protein, binds to sequences within
replicator Interacts with two other proteins –
CDC6 & CDT1 resulting in loading of MCM
complex on DNA .
MCM (Mini chromosome maintenance complex)
Heterohexamer (MCM2-MCM7), ring shaped
replicative helicase, Once the Mcm proteins have
been loaded onto the chromatin, ORC and Cdc6
can be removed from there.
DNA ligase
• They are class of enzymes that catalyze the formation
of alpha phosphodiester bond between two DNA
chains.
• This enzyme bind OH group at the 3' end of DNA
strand to phosphate group at 5' end of the other.
• Linking the two fragments and completing replication
of the region of the lagging strand.
STAGES OF EUKARYOTIC DNA REPLICATION

INITIATION

ELONGATION

TERMINATION
 EUKARYOTIC DNA REPLICATION

❑ During initiation, proteins bind to the origin of replication

while helicase unwinds the DNA helix and two replication
forks are formed at the origin of replication.
❑ During elongation, a primer sequence is added with
complementary RNA nucleotides, which are then replaced
by DNA nucleotides.
❑ During elongation the leading strand is made continuously,
while the lagging strand is made in pieces called Okazaki
fragments.
❑ During termination, primers are removed and replaced with
new DNA nucleotides and the backbone is sealed by DNA
ligase.
 Initiation
To accomplish this, initiation is partitioned into
two temporally discrete steps:

• Pre replication complex (Pre RC) formation

during G1 phase
• Activation of pre RC by cdk and ddk enzymes
in S phase.
 Cell Cycle Regulation
• G1 phase of the cell cycle there are low levels of CDK
activity.
• This allows for the formation of new pre-RC complexes
• During the remaining phases of the cell cycle there are
elevated levels of CDK activity.
• This inhibiting new pre-RC complex formation.
• At the transition of the G1 stage to the S phase of the
cell cycle, S phase–specific cyclin-dependent protein
kinase (CDK) and Dbf4 kinase (DDK) transform the
pre-RC into an active replication fork.
Effect of Cdk Activity on pre-RC Formation
and Activation
Step in the Formation of the pre-RC

• Recognition of the replicator by the

eukaryotic initiator, ORC (Origin
Recognition Complex)
• Once ORC is bound, it recruits two
helicase loading proteins (cell
division cycle 6 protein) and
(chromatin licensing and DNA
replication factor 1 protein).
• ORC and the loading proteins recruits
eukaryotic replication protein i.e.
helicase (the Mcm 2-7 complex)
• Instead the pre-RCs that are formed
during G1 are only activated to initiate
replication after cells pass from the G1
to the S phase of the cell cycle
• Phosphorylation of these proteins
results in the assembly to add
additional replication proteins at the
origin and the initiation of
replication at S phase.
• These new proteins include the
three eukaryotic DNA polymerases.
• Then converted to the active CMG
(Cdc45-MCM-GINS) helicase
during S phase.
• Once DNA Pol α/ primase
synthesizes an RNA primer and
briefly extends it. Thus initiation of
replication started.
• RFC (Replication
Factor C) recognizes
primer-template
junctions and loads
PCNA (Proliferating
Cell Nuclear Antigen)
at these sites.
 Enzymatic activity
• Topoisomerases are responsible for removing
supercoils ahead of the replication fork.
• Helicase unwind the strands at fork.
• Pol α is associated with an RNA primase
synthesizing a primer of 10 nucleotide stretch
of RNA.
• Pol α & δ for priming synthesis & elongates
the newly formed primer with DNA
nucleotides.
The Eukaryotic Replication fork:
• The progress of the replication fork in eukaryotes is
maintain by helicase activity .
• The separated polynucleotides are prevented from
reattaching by SSBs Proteins.
• The main SSBs Protein in eukaryotes is RPA
Elongation
 Leading strand synthesis
• Starts with the primase activity of DNA Pol α to lays down
an RNA primer, then the DNA pol component of Pol α adds
a stretch of DNA.
• RFC assembles PCNA at the end of the primer, PCNA
displaces DNA Pol α.
• DNA polymerase δ or e binds to PCNA at the 3’ ends of
the growing strand to carry out highly processive DNA
synthesis Leading strand synthesis.
• The DNA Polymerase α can extend the initial RNA primer
with about 20 nucleotides of DNA but not capable of
lengthy DNA synthesis.
• After that DNA polymerase δ recognizes this primer and
begins leading strand synthesis in 5′ —> 3′ direction,
Priming DNA Synthesis in Bacteria
& Eukaryotes
 Lagging strand synthesis
• RNA primers synthesized by DNA polymerase α in the 5′ 3′ direction.

• DNA polymerase δ will synthesize short fragments of DNA called Okazaki

fragments which are added to the 3' end of the primer.

• Completion of lagging-strand synthesis requires removal of the RNA

primer from each okazaki fragments.

• To solve this problem there is two models for completion of lagging strand
synthesis in eukaryotes are described:
– The RNase H model

– The flap model

A) RNAse H Model B) Flap Model
• Histone syntesis is continuous threw out the cell
cycle but most amount produced during s phase.

• Protein responsible for that are NAP-1

(nucleosome assembly protein) having a role in
to transport histone from cytoplasm to nucleus.

• Other protein is CAF-1(chromatin assembly

factor) that deliver histone to the site of
replication .
HOW REPLICATION TAKES PLACE AT
TELOMERIC ENDS....?

Termination
• DNA polymerase can not replicate the end of lagging strand

because polymerase requires 3' OH end to bind at daughter

strand. so it require some additional enzyme to perform it.

• TELOMERE is G - C rich terminal portion of chromosome.

• Highly repetitive and conserved sequenced present in eukaryotes.

• TELOMERIC sequence in –

➢ MAMMALS - TTAGGG

➢ ARABIDOPSIS - TTTAGGG

➢ PARAMECIUM -TTGGGG
 Termination of Replication in Eukaryotes
• Removal of the terminal primer at the end of lagging-strand synthesis
leaves a small gap that cannot be filled in, if left unfixed, this gap leads to
the loss of terminal sequences. This is known as the end-replication
problem. Telomerase (telomere terminal transferase) is a ribonucleoprotein
solve this problem

• Telomerase contains protein and RNA which is complementary to the

DNA sequence found in the telomeric repeat. Telomerase extend the 3' end
of the parental chromosome beyond the 5‘ end of the daughter strand.

• Telomeric repeat-binding factor (TRF1) and TRF2 bind to double-stranded

telomeric DNA and have a role in telomere stabilization and telomere-
length regulation. The protruding 3′ end can invade the duplex DNA and
form a lariat-like structure called ‘t-loop’, establishing a protective cap .
 Telomeric replication
• Telomeres are special nucleoprotein structures composed of doublestranded
(TTAGGG)n DNA repetitive sequence ranging from 3 to 15 kb ∼ and a number of
telomere associated proteins.

• The ds telomeric DNA terminates at a 3′ single-stranded G-rich overhang of about

12–500 bases. This protruding 3′ end can invade the duplex DNA and form a lariat-
like structure called ‘tloop’. Establishing a protective cap that shields chromosome
ends from being recognized as damaged DNA and prevents nucleolytic degradation
and inappropriate fusions of telomeres.

• The t-loop is stabilized by a complex of ds and ss stranded telomere binding

proteins known as the ‘shelterin’ proteins [TRF1, TRF2]. The primary role of
shelterin is to mark telomeres as the natural chromosome ends and suppress the
DNA damage response pathways at telomeres
 Telomeric activity and aging
• Telomerase activity is repressed in the somatic
cells of multicellular organism, resulting in a
gradual shortening of the chromosomes with each
cell generation, and ultimately cell death (related
to cell aging).
• This telomerase activity find in germ cells and
absent in somatic cell, except cancerous cell.
• In humans due to shortening of telomeric length
cause a inherited diseases of premature aging
known as progerious occur in teen age.
ROLLING CIRCLE OF
REPLICATION
 INTRODUCTION
• Rolling circle replication is a process of
unidirectional nucleic acid replication.
• Can rapidly synthesize multiple copies of
circular molecules of DNA or RNA, such as
plasmids.
• Eukaryotes also replicate.
• Widely used in molecular biology &
biomedical nanotechnology, especially in the
field of biosensing (as a method of signal
Amplification).
STEPS OF ROLLING CIRCLE REPLICATION

Initiation

Elongation

Termination
STEPS
1) Circular ds DNA will be “nicked”
2) 3` end is elongated →Leading strand
3) 5` end displaced → Lagging strand made up of double
stranded by OKAZAKI fragments.
4)Replication of both “ un-nicked” and displaced ss DNA
5)Displaced DNA circulates and synthesis its own
complementary strand.
→Replication in
eukaryotes is
bidirectional, this type is
Unidirectional.

→Ideal example of this

type is the circular
plasmid is bacteria, as
it happens only in
circular genomes.
Initiation
Initiates - phosphate ends, by the action of:
a) Helicase
b) Topoisomerases
c) Single stranded binding proteins (SSBPs).
Elongation
• For Elongation, - OH group of broken strand, using the
unbroken strand as a template. The polymerase will start
to move in a circle for elongation, due to which it is
named as Rolling Circle Model.
• End will be displaced and will grow out like a waving
thread.
III
Termination
• At the point of termination, the linear DNA molecule is
cleaved from the circle resulting in a double stranded
circular DNA molecule and a single- stranded linear DNA
molecule.
• The linear single stranded molecule is circularized by the
action of ligase and then replication to double stranded
circular plasmid molecule.
BEST EXAMPLE
 SYNTHESIS OF (+) STRAND
• Loop rolling replication.
• Begins with primosome added to gene A.
• Helicase attaches to (-) strand at gene A.
• Pol III extends the (+) strand from 3’-OH.
• Extension process generates a loop rolling
structure.
• Old (+) strand is pelled off.
• Simultaneously newly formed 3’OH of old
looped strand attacks its 5’ phosphate
terminus attaches to 5’protein A.
 SYNTHESIS OF (-) STRAND
• The (+) strand is coated with SSB ,except for PAS.
• This recognized by pri A, pri B, pri C .
• Dna B and Dna C add to the DNA.
• Dna C then releases.
• Primose pro-pelled in 5’-3’direction along with (+)
strand by pri A.
• Pol III extends the primer.
• Pol I excises the primer.
• Fragments are joined by ligase and super coiled
by gyrase.
• Forms (-) strand.
Examples:
• Viral DNA
– Human herpes virus
– Human papilloma virus
– Geminivirus
• Viral RNA
– Pospiviridiae
– Avsunviridiae
Fast & accurate!
Fidelity of replication—a matter of
proofreading
• DNA that is transmitted to daughter cells must be accurately
duplicated to maintain genetic integrity and to promote
genetic continuity.
• A major function of replicative DNA polymerases is to
replicate DNA with the very high accuracy.
• The fidelity of DNA replication relies on
• nucleotide selectivity of replicative DNA polymerase
• exonucleolytic proofreading
• postreplicative DNA mismatch repair (MMR)
• Proofreading activity that assists most of the replicative
polymerases is responsible for removal of incorrectly
incorporated nucleotides from the primer terminus before
further primer extension.
• It is estimated that proofreading improves the fidelity by a 2–
3 orders of magnitude.
 Editing & proofreading DNA

Proofreading refers to any

mechanism for correcting errors
in protein or nucleic acid
synthesis that involves scrutiny
of individual units after they
have been added to the chain

DNA polymerase δ and ε

• proofreads & corrects
mistakes
• repairs mismatched bases
• removes abnormal bases
• repairs damage throughout life
Replicative polymerases use a variety of ways to
accomplish high fidelity DNA replication:
(1) detecting the correct base pair’s suitable
geometry,
(2) slowing down catalysis when a mismatch
occurs, and
(3)partitioning the mismatched primer to the
active site of the exonuclease.
 Check points
• In order to preserve genetic information during cell division, DNA
replication must be completed with high fidelity.

• To achieve this task, eukaryotic cells have proteins in place during

certain points in the replication process that are able to detect any
errors during DNA replication and are able to preserve genomic
integrity.

• These checkpoint proteins inactivate cyclin dependent kinases to

inhibit cell cycle progression from G1 to S.
INHIBITORS OF DNA REPLICATION
CIPROFLOXACIN

NOVOBIOCIN

NALIDIXIC ACID

ADRIYAMYCIN

ETOPOSIDE

DOXORUBICIN
Difference between prokaryotic and eukaryotic DNA replication

Characteristic Prokaryote Eukaryote

Site of DNA replication Cytoplasm Nucleus
Chromosome Circular Linear
No. of origins Single Multiple
Telomeres Absent Present
DNA polymerases DNA pol III DNA pol α, δ, ε, γ
Primase dnaG DNA pol α/ primase
Helicase dnaB MCM proteins (Minichromosome
maintainence)
Sliding clamp Beta protein PCNA (Proliferating Cell Nuclear
Antigens) for DNA pol δ
Clamp loader Gamma complex RF C
SSB proteins SSB RF A
Polymerase coupling Gamma ?
Origin of Replication dnaA ORC