DNA REPLICATION - PART – II
By
Dr. Ichha PuraK
University Professor
Department of Botany
Ranchi Women’s College,Ranchi
http://dripurak.com
http://drichhapurak.webnode.com
6/15/2013DNA REPLICATION PART-II1
DNA REPLICATION PART-II
•REPLICATION FORK
•MECHANISM OF REPLICATION : STEPS
•LEADING AND LAGGING STRANDS
•OKAZAKI FRAGMENTS
•DNA POLYMERASES : PROKARYOTES
•EUKARYOTIC DNA POLYMERASE
•DIFFERENCE IN PROKARYOTIC AND EUKARYOTIC REPLICATION
• TELOMERASE ACTIVITY
•PROOF READING BY DP III
•THE BIOCHEMICAL REACTION
•SPEED OF REPLICATION
6/15/2013DNA REPLICATION PART-II2
3
.
Replication
of leading
strand
occurs
continuously
in 5’  3’
direction of
the new
strand
Replication of
lagging strand
occurs
discontinuously.
Short
DNA fragments
are initially
synthesized and
then ligated
together by
DNA ligase.
REPLICATION FORK
MOUTH
NECK
6/15/2013DNA REPLICATION PART-II4
MECHANISM OF REPLICATION
DNA is replicated by the coordinated efforts of a number of
proteins and enzymes
Mechanism of replication was studied for the first time for the
bacterium Escherichia coli. DNA replication in higher organisms
is less well understood, but involves almost same type of
mechanism with some differences.
For Initiation of DNA Replication ,recognition of Origin of
Replication is required. It is recognized by Pre Priming Complex.
PPC consists of Dna A protein, Single stranded DNA binding
proteins and enzyme Helicase. PPC help in separation of two
parental strands locally and stabilize Replication Fork .
6/15/2013DNA REPLICATION PART-II5
For semi conservative mode of DNA replication the two
strands must get separated by dissolving the hydrogen
bonds between base pairs gradually in a Zipper like manner
and then two separated strands act as templates for
synthesis of new ( daughter ) strands
STEPS OF DNA REPLICATION IN PROKARYOTES
SEPARATION OF TWO PARENTAL STRANDS
1.The two strands separate after formation of Initiation
complex by the enzyme helicase at A-T rich sequences
(Ori).ATP is hydrolysed to provide energy for unwinding. A
single ori is present in prokaryotic circular DNA. Many Ori are
present in Eukaryotic linear DNA. Ori are the consensus
sequences composed almost exclusively of AT base pairs.
SOLVING PROBLEM OF SUPERCOIL FORMATION
2. The opening is facilitated by topoisomerase, which causes a
nick or cut in one of the two strands.Nick is formed by
breaking one phospho di-ester bond near replication fork.The
nick helps to solve the problem of supercoil formation
tendency of DNA double helix on separation of strands.
6/15/2013DNA REPLICATION PART-II6
6/15/2013DNA REPLICATION PART-II7
3. This localized opening appears as bubble under electron
microscope, consisting of two ‘Y’ shaped replication forks.
6/15/2013DNA REPLICATION PART-II8
6/15/2013DNA REPLICATION PART-II9
4.The single stranded DNA structure of replication fork is stabilized by
SSDNA binding proteins which act in co-operative way . Binding of one
SSBp help other SSBPs to bind on both sides of single strands
5. DP III starts synthesis by adding complementary
deoxyribonucleotides onto 3’ OH end of RNA primer
6.A short sequence of Ribonucleotides (RNA Primer) is synthesized
prior to DNA synthesis by primase
ROLE OF RNA PRIMERS IN DNA REPLICATION
DNA Polymerase can not initiate DNA synthesis simply on single stranded
DNA template. They require an RNA Primer ,short oligo ribonucleotide
that makes base pair with few of exposed bases on DNA template having
a free 3’OH group , which acts as first acceptor of incoming
deoxyribonucleotide introduced by DNA polymerase.
6/15/2013DNA REPLICATION PART-II10
PRIMASE (A Specific RNA Polymerase ) synthesizes RNA primer of
about 10 nucleotides long that are complementary and antiparallel to
DNA template. In the resulting hybrid duplex U of RNA pairs with A of
DNA. Only one RNA primer is synthesized in one origin on Leading
strand but many RNA primers are synthesized on Lagging strand
PRIMOSOME
Pre Priming Compex of several proteins is assembled and binds to
single stranded DNA displacing some of SSBPs .This protein complex
along with enzyme Primase is called as Primosome. It initiates Okazaki
Fragment formation by moving along the template for Lagging strand in
the 5’→3’ direction for synthesis of RNA Primers
6/15/2013DNA REPLICATION PART-II11
6/15/2013DNA REPLICATION PART-II12
.
DIRECTION OF REPLICATION
7. AS DNA polymerase can only add deoxyribonucleotides in 5’ 3’
direction and as two strands of DNA double helix run antiparallel, so
synthesis on two strands differ
6/15/2013DNA REPLICATION PART-II13
LEADING STRAND REPLICATION
8.On one of the strand of replication fork running in 3’ 5’
direction towards the fork, DNA is synthesized continuously in
5’3’ direction using only one RNA primer with its free 3’ OH at the
beginning by DP III movement along the template. It is called as
leading strand LAGGING STRAND REPLICATION
9.On other strand of the replication fork running in 5’ 3’ direction away
from the replication fork , DNA is synthesized in a discontinuous manner in
5’ 3’ direction as small stretches . Each time it requires RNA primer with
its free 3’OH for adding dNTPs by movement of DP III along the template.
As a result many RNA-DNA pieces are formed which are termed as
Okazaki Fragments after their discoverer Okazaki (Japanese Molecular
Biologist). This strand is termed as lagging strand as it has to wait for
replication fork to open gradually.
6/15/2013DNA REPLICATION PART-II14
CHAIN ELONGATION
10.Although both strands replicate simultaneously but DNA
synthesis on lagging strand require help of another two enzymes.
RNase dismantles first the primers and then DP I replaces
deoxyribonuleotides to fill the gaps.
11.After that DNA ligase catalyses phophodiester bond formation
between two successive DNA pieces as a result of which
continuous DNA strand is synthesized.
12.As both strands synthesize DNA simultaneously it is presumed
that two molecules of DP III move in opposite directions along their
respective template strands. This is accomplished by having the
lagging strand template looped back on itself. (Fig Next slide)
6/15/2013DNA REPLICATION PART-II15
Replication of the leading and lagging strands in E coli is
accomplished by two DNA Polymerases III working together as part
of a single complex
6/15/2013DNA REPLICATION PART-II16
Replication of the leading and lagging strands in E coli is
accomplished by two DNA Polymerases III working together as
part of a single complex
The two DNA Polymerase III molecules travel together, even
though they are moving towards the opposite ends of their
respective templates. This is accomplished by causing the lagging
strand template to form a loop. The polymerase releases the
lagging strand template when it encounters the previously
synthesized Okazaki fragment. The polymerase that was involved
in the assembly of the previous Okazaki fragment has now
rebound the lagging strand template farther along its length and is
synthesizing DNA onto the end of RNA Primer that has just been
constructed by the primase
6/15/2013DNA REPLICATION PART-II17
6/15/2013DNA REPLICATION PART-II18
LEADING AND LAGGING STRANDS
To catalyse the polymerization reaction, DP enzyme requires all
the four d NTPs, a template strand to copy, a primer containing free
3’ OH to which nucleotides can be added. The primer is required
because DP is unable to initiate the formation of a DNA strand de
novo. Rather, it is capable of adding nucleotides to a 3’OH terminus
of the existing strand. DP is only capable of polymerizing a strand in
5’ 3’ direction. The two new strands are synthesized in opposite
direction and mode of synthesis is quite different.One of newly
synthesized strands ( leading strand) grows towards the replication
fork and is synthesized continuously.The other newly synthesized
strand ( lagging strand ) grows away from the fork and is
synthesized discontinuously
6/15/2013DNA REPLICATION PART-II19
. In bacterial cells, the lagging strand is synthesized as okazaki
fragments approximately 1000 to 2000 nucleotide long. In eukaryotes
also DNA synthesis on lagging strand is discontinuous but the
okazaki fragments are considerably smaller
Okazaki fragments have RNA primers and DNA stretches, For
ligation, first RNA primers are removed by RNases and the gaps are
filled by deoxyribonucleotides by DPI. DNA ligase establishes
phospho - diester bond between two DNA pieces as a result
continuous stretch of new DNA is synthesized on lagging strand
away from the replication fork. Two molecules of DP II are thought to
move together as a complex in opposite directions along their
respective template strands.
6/15/2013DNA REPLICATION PART-II20
OKAZAKI FRAGMENTS
6/15/2013DNA REPLICATION PART-II21
DNA POLYMERASE ENZYME IN PROKARYOTES
In bacterial cell,3 different types of DP are present, although
all of them have the same basic catalytic activity,which is to add
deoxyribonucleotides onto the 3’OH end of a single stranded
primer (short RNA sequences). However they differ in their
various roles within the cell and their molecular structure.
The enzyme that acts in DNA strand formation during
replication in E.coli is DP III and is often called as replicase. DP
III holoenzyme ( core consisting of 3 subunits ,, ) is much
larger than other two polymerases, consisting of a single
catalytic subunit and at least nine different subunits having
various functions.
One of subunit  , appears responsible for keeping DP III
associated with DNA template. DP III has two contrasting
properties. At one end it remains associated with the
template over long stretches to synthesize a continuous
complementary strand. At the same time it has to move from
one nucleotide to other, which is probably helped by its
subunits. The function of DP II is yet unknown. DP I is thought
to be involved primarily in DNA repair , it also removes RNA
primers at the 5’ end of each Okazaki fragment and replaces
them with deoxyribonuleotides.
6/15/2013DNA REPLICATION PART-II22
6/15/2013DNA REPLICATION PART-II23
The DNA polymerase III holoenzyme is a multisubunit complex, which
consists of 17 polypeptides. It contains four sub assemblies. First, the core
polymerase consists of three subunits: α (the polymerase); ε (the 3'–5'
exonuclease); and θ (the stimulator of the 3'–5' exonuclease). Second, the
π subunit is responsible for dimerization of the core DNA polymerase.
Third, the sliding clamp comprises two homo dimers of the β subunit,
which provides the ring structure that is needed for processivity. Fourth,
five subunits have β clamp-loader functions .
6/15/2013DNA REPLICATION PART-II24
6/15/2013DNA REPLICATION PART-II25
EUKARYOTIC DNA POLYMERASE
To date 5 different DNA polymerases have been isolated from
eukaryotic cells and are designated as , , , and . Polymerase  is
tightly associated with the primase, which initiates synthesis of
primers at the 5’ end of each okazaki fragment as the polymerase –
primase complex moves along the lagging strand template.
All the DP require some divalent cat ions as prokaryotic DP
It appears that the leading strand and most of the fragments of the
lagging strand are assembled by DP  ( delta).
DP  replicates mitochondrial DNA and DP  functions in DNA
repair. DP  (epsilon) binds to template of lagging strand and
synthesize discontinuous okazaki fragments. Like prokaryotic
polymerases, all the eukaryotic enzymes elongate DNA strands
in the 5’ 3’ direction by addition of nucleotides to a 3’ OH
group and non of them is able to initiate the synthesis of DNA
chain without a primer. Eukaryotic polymerases posses a 3’ 5’
exonuclease activity ensuring that replication occurs with very
high accuracy.
6/15/2013DNA REPLICATION PART-II26
6/15/2013DNA REPLICATION PART-II27
S
N
Polymerase Function Proof
reading
1 Pol α Contains primase
Initiates DNA
synthesis
-
2 Pol β Repair -
3 Pol γ Replicates
mitochondrial
DNA
+
4 Pol δ Elongates
leading strands
and Okazaki
fragments
+
5 Pol ε Repair +
Table : ACTIVITIES OF EUKARYOTIC
DNA POLYMERASES (Pols)
6/15/2013DNA REPLICATION PART-II28
DIFFERENCE IN PROKARYOTIC AND EUKARYOTIC
REPLICATION
1.As eukaryotic DNA is many times larger than prokaryotic DNA
and is linear, there are multiple origins of replication ( Fig.3).
2.Both in prokaryotes and Eukaryotes replication proceeds bi-
directionally from the origin of replication.
3.In eukaryotes all the origins of replication start DNA synthesis
simultaneously and ultimately meet each other to complete
replication from one end to other.
4.The Okazaki fragments formed in prokaryotes are much larger
(1000-2000 nucleotides long) than that formed in eukaryotes
averaging about 250 nucleotides only in length. All rest of the
steps in eukaryotic replication are same as prokaryotic
6/15/2013DNA REPLICATION PART-II29
6/15/2013DNA REPLICATION PART-II30
Eukaryotic DNA Replication with many origins of replication.
Replication proceeds bi-directionally from each origin
6/15/2013DNA REPLICATION PART-II31
TELOMERASE ACTIVITY
As eukaryotic DNA is linear , it faces problem in replicating
the ends. Folllowing removal of RNA primer from the
extreme 5’ end on lagging strand , there is no way to fill
the gap by deoxyribonucleotides.
To solve this problem and to protect the ends of DNA
(chromosome ) against nucleases , the terminities of DNA
has generally Non coding sequence which are highly
repetitive having T/G . The ends are known as telomeres.
The TG strand is longer than its complement ,leaving a
region of single stranded DNA at the 3’ end of the double
helix that is a few hundred nucleotides long.
6/15/2013DNA REPLICATION PART-II32
RNA of telomerase base pairs with terminal nucleotides at the
single stranded 3’ end of DNA . The RNA then serves as a
template for extending the DNA strand , once the next repeat
sequence is complete, telomerase RNA is translocated to
newly synthesized end of DNA ,where it again hydrogen
bonds and the proccess is completed
Telomerase is a special kind of Reverse Transcriptase that
carries its own RNA molecule of about 150 nucleotide long
having copies of A/C sequence that is complementary to the
T/G repeat sequence
6/15/2013DNA REPLICATION PART-II33
Mechanism of action
of telomerase
Reproduced –from lipincott’s Illustrated
Reviews:Biochemistry
6/15/2013DNA REPLICATION PART-II34
PROOF READING BY DP III
Exact Replication of DNA is partly due to strict base pairing provision and
partly due to proof reading property of DNA Polymerase I & III in 3’ 5’
direction by hydrolyzing the wrong nucleotides.
To ensure Replication fidelity(accuracy), DNA Polymerase III has, in
addition to its 5’3’ polymerase activity , a proof reading activity in
3’5’direction,acts as exonuclease. As each nucleotide is added to the
chain, DP III checks to make sure the added nucleotide is in fact,
correctly matched to its complementary base on the template and edits
its mistakes. For example if the template base adenine and the enzyme by
mistake has introduced cytosine instead of Thymine to the new chain, DP
III hydrolytically removes the mismatched nucleotide and replaces it with
correct nucleotide (T).
6/15/2013DNA REPLICATION PART-II35
1
DPIII inserts correct
nucleotide to its
complementary base on
DNA template and is
added to growing chain
2
Proof reading function of DP
III (3’-------→5’) . If DP III
mispairs a nucleotide on the
template , it utilizes its 3’-------
→5 exonuclease activity to
excise the wrong nucleotide
6/15/2013DNA REPLICATION PART-II36
THE BIOCHEMICAL REACTIONS
DNA replication begins with the "unzipping" of the parent molecule as the
hydrogen bonds between the base pairs are broken.
Once exposed, the sequence of bases on each of the separated strands
serves as a template to guide the insertion of a complementary set of bases
on the strand being synthesized by DNA Polymerase which is Mg ++ ion
dependent
The new strands are assembled from dNTPs The deoxynucleotide 5’-
triphosphate (dNTP) is the reagent for nucleotide incorporation
Each incoming nucleotide is covalently linked to the "free" 3' carbon atom on
the pentose as the second and third phosphates are removed together as a
molecule of pyrophosphate (p-p) .
By release of Pyrophosphate energy is liberated which helps in formation of
Phoshpho di ester bond with subsequent nucleotide.
The nucleotides are assembled in the order that complements the order
of bases on the strand serving as the template.
Thus each C on the template guides the insertion of a G on the new
strand, each G a C, and so on.
When the process is complete, two DNA molecules have been formed
identical to each other and to the parent molecule.
6/15/2013DNA REPLICATION PART-II37
G
O
O
O
P
O
-O
A
O
O
O
T
O
OH
O P O
O
C
O
OH
O
O-
P
O-
O
O P
O-
O
O-
Mg2+
5'
3'
5'
template
strand
(old)
(new)
3’-hydroxyl group of the
growing DNA strand acts as a
nucleophile and attacks the
-phosphorus atom of the
dNTP.
dNTP
6/15/2013DNA REPLICATION PART-II38
DNA SYNTHESIS : PHOSPHO-DIESTER BOND FORMATION
6/15/2013DNA REPLICATION PART-II39
USE OF RNA PRIMER
TO INITIATE DNA
SYNTHESIS
6/15/2013DNA REPLICATION PART-II40
6/15/2013DNA REPLICATION PART-II41
SPEED OF REPLICATION
Prokaryotes : Genome size of E.coli is 4.7 x 106 nucleotide
pairs. Replication proceeds from single origin of replication at
the speed of 1000 nucleotides per sec completing replication
of whole genome in 40 minutes.
Eukaryotes : Speed is very slow in comparision to
prokaryotes. The average human chromosome contains 150x
106 nucleotide pairs which are copied at about only 50 bp per
sec. But because there are multiple origins of replication
present along the linear DNA, it is completed in a hour.
6/15/2013DNA REPLICATION PART-II42

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Dna replication part ii

  • 1. DNA REPLICATION - PART – II By Dr. Ichha PuraK University Professor Department of Botany Ranchi Women’s College,Ranchi http://dripurak.com http://drichhapurak.webnode.com 6/15/2013DNA REPLICATION PART-II1
  • 2. DNA REPLICATION PART-II •REPLICATION FORK •MECHANISM OF REPLICATION : STEPS •LEADING AND LAGGING STRANDS •OKAZAKI FRAGMENTS •DNA POLYMERASES : PROKARYOTES •EUKARYOTIC DNA POLYMERASE •DIFFERENCE IN PROKARYOTIC AND EUKARYOTIC REPLICATION • TELOMERASE ACTIVITY •PROOF READING BY DP III •THE BIOCHEMICAL REACTION •SPEED OF REPLICATION 6/15/2013DNA REPLICATION PART-II2
  • 3. 3 . Replication of leading strand occurs continuously in 5’  3’ direction of the new strand Replication of lagging strand occurs discontinuously. Short DNA fragments are initially synthesized and then ligated together by DNA ligase. REPLICATION FORK MOUTH NECK
  • 4. 6/15/2013DNA REPLICATION PART-II4 MECHANISM OF REPLICATION DNA is replicated by the coordinated efforts of a number of proteins and enzymes Mechanism of replication was studied for the first time for the bacterium Escherichia coli. DNA replication in higher organisms is less well understood, but involves almost same type of mechanism with some differences. For Initiation of DNA Replication ,recognition of Origin of Replication is required. It is recognized by Pre Priming Complex. PPC consists of Dna A protein, Single stranded DNA binding proteins and enzyme Helicase. PPC help in separation of two parental strands locally and stabilize Replication Fork .
  • 5. 6/15/2013DNA REPLICATION PART-II5 For semi conservative mode of DNA replication the two strands must get separated by dissolving the hydrogen bonds between base pairs gradually in a Zipper like manner and then two separated strands act as templates for synthesis of new ( daughter ) strands STEPS OF DNA REPLICATION IN PROKARYOTES SEPARATION OF TWO PARENTAL STRANDS 1.The two strands separate after formation of Initiation complex by the enzyme helicase at A-T rich sequences (Ori).ATP is hydrolysed to provide energy for unwinding. A single ori is present in prokaryotic circular DNA. Many Ori are present in Eukaryotic linear DNA. Ori are the consensus sequences composed almost exclusively of AT base pairs.
  • 6. SOLVING PROBLEM OF SUPERCOIL FORMATION 2. The opening is facilitated by topoisomerase, which causes a nick or cut in one of the two strands.Nick is formed by breaking one phospho di-ester bond near replication fork.The nick helps to solve the problem of supercoil formation tendency of DNA double helix on separation of strands. 6/15/2013DNA REPLICATION PART-II6
  • 7. 6/15/2013DNA REPLICATION PART-II7 3. This localized opening appears as bubble under electron microscope, consisting of two ‘Y’ shaped replication forks.
  • 9. 6/15/2013DNA REPLICATION PART-II9 4.The single stranded DNA structure of replication fork is stabilized by SSDNA binding proteins which act in co-operative way . Binding of one SSBp help other SSBPs to bind on both sides of single strands 5. DP III starts synthesis by adding complementary deoxyribonucleotides onto 3’ OH end of RNA primer 6.A short sequence of Ribonucleotides (RNA Primer) is synthesized prior to DNA synthesis by primase ROLE OF RNA PRIMERS IN DNA REPLICATION DNA Polymerase can not initiate DNA synthesis simply on single stranded DNA template. They require an RNA Primer ,short oligo ribonucleotide that makes base pair with few of exposed bases on DNA template having a free 3’OH group , which acts as first acceptor of incoming deoxyribonucleotide introduced by DNA polymerase.
  • 10. 6/15/2013DNA REPLICATION PART-II10 PRIMASE (A Specific RNA Polymerase ) synthesizes RNA primer of about 10 nucleotides long that are complementary and antiparallel to DNA template. In the resulting hybrid duplex U of RNA pairs with A of DNA. Only one RNA primer is synthesized in one origin on Leading strand but many RNA primers are synthesized on Lagging strand PRIMOSOME Pre Priming Compex of several proteins is assembled and binds to single stranded DNA displacing some of SSBPs .This protein complex along with enzyme Primase is called as Primosome. It initiates Okazaki Fragment formation by moving along the template for Lagging strand in the 5’→3’ direction for synthesis of RNA Primers
  • 12. 6/15/2013DNA REPLICATION PART-II12 . DIRECTION OF REPLICATION 7. AS DNA polymerase can only add deoxyribonucleotides in 5’ 3’ direction and as two strands of DNA double helix run antiparallel, so synthesis on two strands differ
  • 13. 6/15/2013DNA REPLICATION PART-II13 LEADING STRAND REPLICATION 8.On one of the strand of replication fork running in 3’ 5’ direction towards the fork, DNA is synthesized continuously in 5’3’ direction using only one RNA primer with its free 3’ OH at the beginning by DP III movement along the template. It is called as leading strand LAGGING STRAND REPLICATION 9.On other strand of the replication fork running in 5’ 3’ direction away from the replication fork , DNA is synthesized in a discontinuous manner in 5’ 3’ direction as small stretches . Each time it requires RNA primer with its free 3’OH for adding dNTPs by movement of DP III along the template. As a result many RNA-DNA pieces are formed which are termed as Okazaki Fragments after their discoverer Okazaki (Japanese Molecular Biologist). This strand is termed as lagging strand as it has to wait for replication fork to open gradually.
  • 14. 6/15/2013DNA REPLICATION PART-II14 CHAIN ELONGATION 10.Although both strands replicate simultaneously but DNA synthesis on lagging strand require help of another two enzymes. RNase dismantles first the primers and then DP I replaces deoxyribonuleotides to fill the gaps. 11.After that DNA ligase catalyses phophodiester bond formation between two successive DNA pieces as a result of which continuous DNA strand is synthesized. 12.As both strands synthesize DNA simultaneously it is presumed that two molecules of DP III move in opposite directions along their respective template strands. This is accomplished by having the lagging strand template looped back on itself. (Fig Next slide)
  • 15. 6/15/2013DNA REPLICATION PART-II15 Replication of the leading and lagging strands in E coli is accomplished by two DNA Polymerases III working together as part of a single complex
  • 16. 6/15/2013DNA REPLICATION PART-II16 Replication of the leading and lagging strands in E coli is accomplished by two DNA Polymerases III working together as part of a single complex The two DNA Polymerase III molecules travel together, even though they are moving towards the opposite ends of their respective templates. This is accomplished by causing the lagging strand template to form a loop. The polymerase releases the lagging strand template when it encounters the previously synthesized Okazaki fragment. The polymerase that was involved in the assembly of the previous Okazaki fragment has now rebound the lagging strand template farther along its length and is synthesizing DNA onto the end of RNA Primer that has just been constructed by the primase
  • 18. 6/15/2013DNA REPLICATION PART-II18 LEADING AND LAGGING STRANDS To catalyse the polymerization reaction, DP enzyme requires all the four d NTPs, a template strand to copy, a primer containing free 3’ OH to which nucleotides can be added. The primer is required because DP is unable to initiate the formation of a DNA strand de novo. Rather, it is capable of adding nucleotides to a 3’OH terminus of the existing strand. DP is only capable of polymerizing a strand in 5’ 3’ direction. The two new strands are synthesized in opposite direction and mode of synthesis is quite different.One of newly synthesized strands ( leading strand) grows towards the replication fork and is synthesized continuously.The other newly synthesized strand ( lagging strand ) grows away from the fork and is synthesized discontinuously
  • 19. 6/15/2013DNA REPLICATION PART-II19 . In bacterial cells, the lagging strand is synthesized as okazaki fragments approximately 1000 to 2000 nucleotide long. In eukaryotes also DNA synthesis on lagging strand is discontinuous but the okazaki fragments are considerably smaller Okazaki fragments have RNA primers and DNA stretches, For ligation, first RNA primers are removed by RNases and the gaps are filled by deoxyribonucleotides by DPI. DNA ligase establishes phospho - diester bond between two DNA pieces as a result continuous stretch of new DNA is synthesized on lagging strand away from the replication fork. Two molecules of DP II are thought to move together as a complex in opposite directions along their respective template strands.
  • 21. 6/15/2013DNA REPLICATION PART-II21 DNA POLYMERASE ENZYME IN PROKARYOTES In bacterial cell,3 different types of DP are present, although all of them have the same basic catalytic activity,which is to add deoxyribonucleotides onto the 3’OH end of a single stranded primer (short RNA sequences). However they differ in their various roles within the cell and their molecular structure. The enzyme that acts in DNA strand formation during replication in E.coli is DP III and is often called as replicase. DP III holoenzyme ( core consisting of 3 subunits ,, ) is much larger than other two polymerases, consisting of a single catalytic subunit and at least nine different subunits having various functions.
  • 22. One of subunit  , appears responsible for keeping DP III associated with DNA template. DP III has two contrasting properties. At one end it remains associated with the template over long stretches to synthesize a continuous complementary strand. At the same time it has to move from one nucleotide to other, which is probably helped by its subunits. The function of DP II is yet unknown. DP I is thought to be involved primarily in DNA repair , it also removes RNA primers at the 5’ end of each Okazaki fragment and replaces them with deoxyribonuleotides. 6/15/2013DNA REPLICATION PART-II22
  • 24. The DNA polymerase III holoenzyme is a multisubunit complex, which consists of 17 polypeptides. It contains four sub assemblies. First, the core polymerase consists of three subunits: α (the polymerase); ε (the 3'–5' exonuclease); and θ (the stimulator of the 3'–5' exonuclease). Second, the π subunit is responsible for dimerization of the core DNA polymerase. Third, the sliding clamp comprises two homo dimers of the β subunit, which provides the ring structure that is needed for processivity. Fourth, five subunits have β clamp-loader functions . 6/15/2013DNA REPLICATION PART-II24
  • 25. 6/15/2013DNA REPLICATION PART-II25 EUKARYOTIC DNA POLYMERASE To date 5 different DNA polymerases have been isolated from eukaryotic cells and are designated as , , , and . Polymerase  is tightly associated with the primase, which initiates synthesis of primers at the 5’ end of each okazaki fragment as the polymerase – primase complex moves along the lagging strand template. All the DP require some divalent cat ions as prokaryotic DP It appears that the leading strand and most of the fragments of the lagging strand are assembled by DP  ( delta).
  • 26. DP  replicates mitochondrial DNA and DP  functions in DNA repair. DP  (epsilon) binds to template of lagging strand and synthesize discontinuous okazaki fragments. Like prokaryotic polymerases, all the eukaryotic enzymes elongate DNA strands in the 5’ 3’ direction by addition of nucleotides to a 3’ OH group and non of them is able to initiate the synthesis of DNA chain without a primer. Eukaryotic polymerases posses a 3’ 5’ exonuclease activity ensuring that replication occurs with very high accuracy. 6/15/2013DNA REPLICATION PART-II26
  • 27. 6/15/2013DNA REPLICATION PART-II27 S N Polymerase Function Proof reading 1 Pol α Contains primase Initiates DNA synthesis - 2 Pol β Repair - 3 Pol γ Replicates mitochondrial DNA + 4 Pol δ Elongates leading strands and Okazaki fragments + 5 Pol ε Repair + Table : ACTIVITIES OF EUKARYOTIC DNA POLYMERASES (Pols)
  • 28. 6/15/2013DNA REPLICATION PART-II28 DIFFERENCE IN PROKARYOTIC AND EUKARYOTIC REPLICATION 1.As eukaryotic DNA is many times larger than prokaryotic DNA and is linear, there are multiple origins of replication ( Fig.3). 2.Both in prokaryotes and Eukaryotes replication proceeds bi- directionally from the origin of replication. 3.In eukaryotes all the origins of replication start DNA synthesis simultaneously and ultimately meet each other to complete replication from one end to other. 4.The Okazaki fragments formed in prokaryotes are much larger (1000-2000 nucleotides long) than that formed in eukaryotes averaging about 250 nucleotides only in length. All rest of the steps in eukaryotic replication are same as prokaryotic
  • 30. 6/15/2013DNA REPLICATION PART-II30 Eukaryotic DNA Replication with many origins of replication. Replication proceeds bi-directionally from each origin
  • 31. 6/15/2013DNA REPLICATION PART-II31 TELOMERASE ACTIVITY As eukaryotic DNA is linear , it faces problem in replicating the ends. Folllowing removal of RNA primer from the extreme 5’ end on lagging strand , there is no way to fill the gap by deoxyribonucleotides. To solve this problem and to protect the ends of DNA (chromosome ) against nucleases , the terminities of DNA has generally Non coding sequence which are highly repetitive having T/G . The ends are known as telomeres. The TG strand is longer than its complement ,leaving a region of single stranded DNA at the 3’ end of the double helix that is a few hundred nucleotides long.
  • 32. 6/15/2013DNA REPLICATION PART-II32 RNA of telomerase base pairs with terminal nucleotides at the single stranded 3’ end of DNA . The RNA then serves as a template for extending the DNA strand , once the next repeat sequence is complete, telomerase RNA is translocated to newly synthesized end of DNA ,where it again hydrogen bonds and the proccess is completed Telomerase is a special kind of Reverse Transcriptase that carries its own RNA molecule of about 150 nucleotide long having copies of A/C sequence that is complementary to the T/G repeat sequence
  • 33. 6/15/2013DNA REPLICATION PART-II33 Mechanism of action of telomerase Reproduced –from lipincott’s Illustrated Reviews:Biochemistry
  • 34. 6/15/2013DNA REPLICATION PART-II34 PROOF READING BY DP III Exact Replication of DNA is partly due to strict base pairing provision and partly due to proof reading property of DNA Polymerase I & III in 3’ 5’ direction by hydrolyzing the wrong nucleotides. To ensure Replication fidelity(accuracy), DNA Polymerase III has, in addition to its 5’3’ polymerase activity , a proof reading activity in 3’5’direction,acts as exonuclease. As each nucleotide is added to the chain, DP III checks to make sure the added nucleotide is in fact, correctly matched to its complementary base on the template and edits its mistakes. For example if the template base adenine and the enzyme by mistake has introduced cytosine instead of Thymine to the new chain, DP III hydrolytically removes the mismatched nucleotide and replaces it with correct nucleotide (T).
  • 35. 6/15/2013DNA REPLICATION PART-II35 1 DPIII inserts correct nucleotide to its complementary base on DNA template and is added to growing chain 2 Proof reading function of DP III (3’-------→5’) . If DP III mispairs a nucleotide on the template , it utilizes its 3’------- →5 exonuclease activity to excise the wrong nucleotide
  • 36. 6/15/2013DNA REPLICATION PART-II36 THE BIOCHEMICAL REACTIONS DNA replication begins with the "unzipping" of the parent molecule as the hydrogen bonds between the base pairs are broken. Once exposed, the sequence of bases on each of the separated strands serves as a template to guide the insertion of a complementary set of bases on the strand being synthesized by DNA Polymerase which is Mg ++ ion dependent The new strands are assembled from dNTPs The deoxynucleotide 5’- triphosphate (dNTP) is the reagent for nucleotide incorporation Each incoming nucleotide is covalently linked to the "free" 3' carbon atom on the pentose as the second and third phosphates are removed together as a molecule of pyrophosphate (p-p) . By release of Pyrophosphate energy is liberated which helps in formation of Phoshpho di ester bond with subsequent nucleotide.
  • 37. The nucleotides are assembled in the order that complements the order of bases on the strand serving as the template. Thus each C on the template guides the insertion of a G on the new strand, each G a C, and so on. When the process is complete, two DNA molecules have been formed identical to each other and to the parent molecule. 6/15/2013DNA REPLICATION PART-II37 G O O O P O -O A O O O T O OH O P O O C O OH O O- P O- O O P O- O O- Mg2+ 5' 3' 5' template strand (old) (new) 3’-hydroxyl group of the growing DNA strand acts as a nucleophile and attacks the -phosphorus atom of the dNTP. dNTP
  • 38. 6/15/2013DNA REPLICATION PART-II38 DNA SYNTHESIS : PHOSPHO-DIESTER BOND FORMATION
  • 39. 6/15/2013DNA REPLICATION PART-II39 USE OF RNA PRIMER TO INITIATE DNA SYNTHESIS
  • 41. 6/15/2013DNA REPLICATION PART-II41 SPEED OF REPLICATION Prokaryotes : Genome size of E.coli is 4.7 x 106 nucleotide pairs. Replication proceeds from single origin of replication at the speed of 1000 nucleotides per sec completing replication of whole genome in 40 minutes. Eukaryotes : Speed is very slow in comparision to prokaryotes. The average human chromosome contains 150x 106 nucleotide pairs which are copied at about only 50 bp per sec. But because there are multiple origins of replication present along the linear DNA, it is completed in a hour.