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REPLICATION OF DNA
INDRODUCTION
REQUIREMENTS FOR REPLICATION
DNA Synthesis Requires Deoxynucleoside Triphosphates and a Primer:Template
Junction
Two key substrates must be present, First, new synthesis requires the four deoxynucleoside
triphosphates—dGTP, dCTP, dATP, and Dttp
...
The phosphoryl group proximal to the deoxyribose is called the aphosphate, the middle and distal groups are called the b-phosphate and the g-phosphate
...

The template provides the ssDNA that directs the addition of each complementary
deoxynucleotide
...


primer: template junction is a substrate for DNA synthesis because only the primer is
chemically modified during DNA synthesis
...


DNA Is Synthesized by Extending the 30 End of the Primer
FORMATION OF PHOSPHODIESTER BOND
The new chain grows by extending the 30 end of the primer
...
The phosphodiester bond is formed in an SN2 reaction in which the
hydroxyl group at the 30 end of the primer strand attacks the a-phosphoryl group of the
incoming nucleoside triphosphate
...

The two strands of the double helix have an antiparallel orientation
...


Hydrolysis of Pyrophosphate Is the Driving Force for DNA Synthesis
...


Then result of nucleotide addition and pyrophosphate hydrolysis is the breaking of two highenergy phosphate bonds
...

DNA polymerase uses as single active site to catalyze the addition of any of the four
deoxynucleoside triphosphates
...

The DNA polymerase monitors the ability of the incoming nucleotide to for man A:T or G:C
base pair, rather than detecting the exact nucleotide that enters the active site
...

Incorrect base pairing leads to dramatically lower rates of nucleotide addition as a result of a
catalytically unfavorable alignment of these substrates
...
Although rNTPs are present at
approximately 10-fold higher concentration in the cell, they are incorporated at a rate that is
more than 1000-fold lower than dNTPs
TYPE OF DNA POLYMERASE
3-5 synthesis

3-5 exonuclease activity

5-3 exonuclease activity

DNA POLY 1
DNA POLY 2

no

DNA POLY 3

no

DNA POLY 4

no

no

DNA POLY 5

no

no

STRUCTURE OF DNA Polymerases
Based on the hand analogy, the three domains ofthe polymerase are called the thumb, fingers,
and palm
...
In particular, this region of DNA polymerase binds two divalent metal ions (typically
Mg+2+orZn2+)that alter the chemical environment around the correctly base-paired dNTP and
the 3-OH of the primer
...
This generates a O2 that is
primed for the nucleophilic attack of the a-phosphate of the in coming DNTP
...

This region of the polymerase makes extensive hydrogen-bond contacts with base pairs in the
minor groove of the newly synthesized DNA
...

Finger domain moves to enclose the dNTP
...

The combination of the slowed catalysis and reduced affinity for newly synthesized
mismatched DNA allows the release of the primer strand from the polymerase active site, and
in many cases, this strand binds and is acted on by a proofreading nuclease that removes the
mismatched DNA
...


This bend serves to expose only the first template base after the primer at the catalytic site
and avoids any confusion concerning which

template base should pair with then ext

nucleotide to be added (Fig
...
In contrast to the fingers and the palm,
The thumb domain is not intimately involved in catalysis
...

This serves two purposes
...
Second, the thumb helps to maintain a strong association between the DNA polymerase
and its substrate
...
DNA polymerases are capable of adding as many as
1000 nucleotides/sec to a primer strand
...


Processivity is a characteristic of enzymes that operate on polymeric substrates
...

The rate of DNA synthesis is dramatically increased by adding multiple nucleotides per
binding event
...
Inatypical DNA polymerase reaction, it takes 1 sec for the
DNA polymerase to locate and bind a primer: template junction
...
Once
bound to a primer:template junction, DNA polymerase interacts tightly with much of the
double-stranded portion of the DNA in a sequence-nonspecific manner
...

The sequence-independent nature of these interactions permits the easy movement of the
DNA even after it binds to polymerase
...

Initially, the presence of a 30 exonuclease as part of the same polypeptide as a DNA
polymerase made little sense
...
In the rare event that an in
correct nucleotide is added to the primer strand, the exonuclease removes this nucleotide
from the 30 end of the primer strand
...

The removal of mismatched nucleotides is facilitated by the reduced ability of DNA
polymerase to add a nucleotide adjacent to an incorrectly base paired primer
...

This altered geometry reduces the rate of nucleotide addition in much the same way that
addition of an incorrectly paired dNTP reduces catalysis
...

In essence,proof reading exonucleases work like a “delete key,” removing only the most
recent errors
...

On average, DNA polymerase inserts one incorrect nucleotide for every 105 nucleotides
added
...
This error rate is still significantly short of the actual rate of
mutation observed in a typical cell
the rare event that an incorrect nucleotide is added to the primer strand, the exonuclease
removes this nucleotide from the 30 end of the primer strand
...

As for processive DNA synthesis,proofreading occurs without releasing the DNA from the
polymerase
THE REPLICATION FORK
The junction between the newly separated template strands and the unreplicated duplex DNA
is known as the replication fork
...
The antiparallel nature of DNA creates a

complication for the simultaneous replication of the two exposed templates at the replication
fork
...
On this template
strand, the polymerase simply “chases” the moving replication fork
...
Synthesis of the new
DNA strand directed by the other ssDNA templateis more complicated
...

The new DNA strand directed by this template is known as the lagging strand
...

ENZYMES
Helicase

FUNCTIONS
DNA helicases, catalyze the separation of the two strands of duplex
DNA
...

Typically, DNA helicases that act at replication forks are hexameric
proteins that assume the shape of a ring
...

DNA helicases act processively
...


Single stranded To stabilize the separated strands, ssDNA-binding proteins (SSBs)
DNA
binding rapidly bind to the separated strands
...

SSBs primarily contact ssDNA through electrostatic interactions with the
phosphate backbone and stacking interactions with the DNA bases
...


Topoisomerase

Primase

RNase H

DNA ligase

The supercoils introduced by the action of the DNA helicase are removed
by topoisomerases that act on the unreplicated dsDNA in front of the
replication fork
...
This action
relievesthe accumulation of supercoils
...

DNA polymerase can only efficiently extend 3’-OH primers annealed to
ssDNA templates
...

To replace the RNA primers with DNA, an enzyme called RNase H
recognizes and removes most of each RNA primer
...
RNase H removes all of
the RNA primer except the ribonucleotide directly linked to the DNA
end
...
The final ribonucleotide is removed by a 50 exonuclease
that degrades RNA or DNA from their 50 ends
...
913)
...
This “nick” in the DNA can be repaired by an enzyme
called DNA ligase
...


PROCESS OD DNA REPLICATON
•

Binding of Helicase on DNA strand and responsible to break the hydrogen bond in
dsDNA
...


•

Binding of topoisomerase enzyme to release the stress on strand
...
One key to the high processivity of the DNA
polymerases that act at replication forks is their association with proteins called sliding
DNA clamps
...
The hole in the center of the clamp is
large enough to encircle the DNA double helix and leave room for a layer of one or two
water molecules between the DNA and the protein
...
Importantly, sliding DNA clamps also bind tightly to DNA polymerases bound to
primer: template junctions (Fig
...
The resulting complex between the polymerase
and the sliding clamp moves efficiently along the DNA template during DNA synthesis
...
A special class of protein complexes, called sliding clamp loaders, catalyze the
opening and placement of sliding clamps on the DNA
...
In the presence of ATP sliding clamp open
and in the absence of ATP it remains close
...
Coli (PROKARYOTES)
There are 3 major steps involved in replication
...
They defined all of the DNA replicated
from a particular origin of replication as a replicon
...
coli cells has only one origin of replication, the entire chromosome
is a single replicon
...
coli chromosome replicates by the bidirectional mode from a
single replication origin
...
coli at 37°C varies with growth conditions from
20 min to 10 h
...
6 106-bpE
...
The second component of the replicon model is the initiator protein
...

The single replicator required for E
...
Two
repeated motifs are critical for oriC function (Fig
...
The 9-mer motif is the binding site
for the E
...

The 13-mer motif, repeated three times, is the initial site of ssDNA formation during
initiation
...
And also have helicase activity
...

Due to the presence of DNase B and C helicase is recruited and this helicase responsible to
break the hydrogen bond
...

Once the holoenzyme is present, sliding clamps are assembled on the RNA primers, and the
leading-strand polymerases are engaged
...


TERMINATION
The E
...
30-36;note that oriC is directly opposite the
terminus region on the E
...

The arrest of replication fork motion at Ter sites requires the action of Tus protein,a 309residue monomer that is the product of the tus gene (for terminator utilization substance)
...

A 5-bp segment of the DNA near the side of Tus that permits the passage of the replication
fork (the lower side of Fig
...

As two oppositely moving replication forks collide at the termination site,the newly
synthesized strands become covalently linked to yield two covalently closed double stranded
chromosomes
...

Catenane is the general term for two circles that are linked (similar to links in a chain)
...
”This separation is accomplished by the
action of type II topoisomerases
...
This
reaction can easily decatenate the two circular daughter chromosomes by breaking one DNA
circle and passing the second through the break, allowing their segregation into separate cells
...
As the lagging-strand replication machinery reaches the end of
the chromosome, at some point, primase no longer has sufficient space to synthesize a new
RNAprimer
...
When this DNA product is replicated in the next round, one
of the two products will be shortened and will lack the region that was not fully copied in the
previous round of replication
...
Some
eukaryotic viruses also replicate their DNA via the rolling circle mechanism
...


coli DNA polymerase or analogous enzymes
...
We imagine that the positive strand
is opened, that the newly exposed 5′ end is attached to the ‘membrane,’ and that a
new copy of this strand is synthesized by chain elongation of the 3′ end of the old
strand
...
DNA will be "nicked"
...
The 3' end is elongated using "unnicked" DNA as leading strand (template); 5'
end is displaced
...
Displaced DNA is a lagging strand and is made double stranded via a series
of Okazaki fragments
...
Replication of both "unnicked" and displaced DNA completes
...
Displaced DNA circularizes
...
The initiator protein remains bound to the 5'
phosphate end of the nicked strand, and the free 3' hydroxyl end is released to serve as
a primer for DNA synthesis by DNA polymerase III
...
There is a specific
protein which is called Rep A protein
...
And creat
free 5’P and 3’OH site,
STEP 2 (HELICASE AND DNA POLYMERASE) START REPLICATION
Which is followed by the addition of DNA polymerase III and it recruit the helicase protein
to unwind the DNA strand by the breaking of hydrogen bond
...
And at this time single stranded DNA are get binded with the single
strand binding protein to maintain the single strand of DNA
...

STEP 3 (DNA LIGASE)
DNA ligase are responsible to seals the nick in double strand molecule
...
Then to replicate first ssDNA become lose
and RNA primer get bind on it
...
After completion DNA ligase join the two ends of the
DNA and for double strand DNA
...


THETA MODEL OF REPLICATION
A Theta structure is an intermediate structure formed during the replication of a
circular DNA molecule (prokaryote DNA), two replication forks can proceed independently
around the DNA ring and when viewed from above it resembles the Greek letter "theta" (θ)
...


Process of DNA replication (Theta model) –
•

Initiation of replication occurs at a specific region called origin of replication where the
ds-DNA denatures to form ss-DNA and within which replication commences

•

The locally denatured segment of DNA is called the replication bubble and the 2 strands
in this region using which new complimentary strands are synthesized are called
the template strands

•

As the DNA unwinds, a y-shaped structure is formed at either ends of the replication
bubble
...
In such cases, bidirectional replication occurs

•

The fork is generated by a complex of 7 proteins called primasome that includes – Dna G
primase, Dna B helicase, Dna C helicase assistant, Dna T, Primase A, B and C

•

In coli, the OriC region spans 245 bp and contains clusters of 3 copies of 13-mer and 4
copies of 9-mer sequences

•

To initiate replication, an initiator protein called Dna A ATP (encoded by dna A gene)
binds to 9-mer sequences and denatures the region connecting it to 13-mer sequences by
breaking A-T bonds which are weaker as they are held by 2 hydrogen bonds
...
This forms the initial complex

•

DNA helicase (Dna B) is loaded onto the DNA strands by a helicase loader (Dna C)
...
This forms the pre-priming complex

•

SSBP (Single-stranded Binding Protein) binds the open strands of DNA to prevent
rewinding
...
This happens because DNA
polymerase III can only add nucleotides but cannot initiate synthesis of a new strand
...
The DNA pol III enzyme tethers itself to the ss-DNA via its core
enzyme
...
The strand
that is being synthesized in this direction continuously is called the leading strand while
the strand that is synthesized in the opposite direction is called lagging strand

•

The leading strand requires just 1 primer whereas the lagging strand requires many such
primers
...
After the strands have been
completely synthesized, these okazaki fragments are joined together
...
DNA pol I removes the primers by its 5’ to 3’ exonuclease
activity exposing the template nucleotides
...
This
is known as nick translation

•

Any errors in base-pairing is removed by DNA polymerase III by its 3’ to 5’ exonuclease
activity immediately before proceeding onto the next nucleotide
...
Tus
proteins (Terminus Utilization Substance) bind to ter sites and halt progression of forks
...
Topoisomerase II resolves this problem by breaking some bonds in
DNA molecules so as to separate the strands – Decatenation

Theta model of prokaryotic replication
DNA Replication by Rolling Circle Model –
•

This occurs when a circular ds-DNA genome needs to be made in multiple copies such as
in lambda phage

•

A nick is made at the origin of replication on the outer strand, also called the (+) strand,
making 2 ends of the (+) strand – 5’ and 3’ end

•

The 3’-OH end is extended by replication enzymes which is the leading strand using the
inner or (-) strand as the template
...
Concatamers are formed

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