DNA Replication: From Basics to Lab Synthesis

By Sawsan sajid

DNA replication

 is the process of producing two identical

copies from one original 

DNA

 molecule. This biological

process occurs in all 

living organisms . It 

is the basis

for 

biological inheritance

. DNA is composed from  two

strands and each strand of the original DNA molecule serves

as 

template

 for the production of the complementary strand,

a process referred to as 

semiconservative replication 

and the

process followed by 

proofreading

 or error-checking

mechanisms  to ensure correct reading to the genetic code,.

like all biological polymerization 

processes(Transcription

and Translation

), the process involve 3 stages :

  1- initiation

2-elongation and 3- termination

.

Early studies of DNA replication mainly depend on the

observation of Meselson and Stahl(1958)  ,the British

biochemist John Carnis (1963) and the Japanese scientist

Reiji Okazaki (1968)

Prokaryotic and eukaryotic DNA replication is bidirectional

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Replication in eukaryotic cell start from more than one 

ori c(each

300bp there is oric ) 

 so multiple replication bubbles will form

thus replication here is faster than 

prokaryotic cell

DNA COULD BE SYNTHESISED IN LAB

The dream become true for synthesizing part of the genome

in lab after 3 decade from discovering DNA polymerase

enzyme precisely in 1983 by Kary  mullis who found a genius

way to amplify (

تكثير 

)  any part of the genomic DNA by 

PCR

process (polymerase chain reaction)

the process require the

following:



Template DNA (genomic animal or plant cell , plasmid,

cosmid, bacterial/yeast colony, etc.)



primers :usually forward and reverse  

DNA  primers(17-25bp)

forwarded  from 5 ́→ OH 3́  with  free end thus DNA polymerase

will use this end to add nucleotide to the newly formed strand .in

nature this segment is synthesized by primase enzyme (

RNA

rather than DNA as will discussed latter

) 



buffer  for  DNA  polymerase enzyme  



To enhance enzyme activity we add MgCl

2

  or MgSO4.



dNTPs :The four type is used (dATP, d TTP, d GTP, d CTP) .



Taq  DNA polymerase: heat stable  enzyme is used here . Cos

of its stability in heat during denarturation  step  (95C

º)

Polymerase chain reaction



Polymerase chain reaction

Researchers commonly replicate DNA 

in vitro

 using

the 

polymerase chain reaction

 (PCR). PCR uses a pair

of

 primers

 to span a target region in template DNA, and

then polymerizes partner strands in each direction from

these primers using a thermostable

 Taq DNA polymerase

.

Repeating this process through multiple cycles produces

amplification of the targeted DNA region. At the start of

each cycle, the mixture of template and primers is heated,

separating the newly synthesized molecule and template.

Then, as the mixture cools, both of these become templates

for annealing of new primers, and the polymerase extends

from these. As a result, the number of copies of the target

region doubles each round, 

increasing exponentially

Properties of Taq DNA polymerase

Taq

 polymerase  is a thermostable DNA polymerase named after

the thermophilic bacterium 

Thermus aquaticus

 from which it was

originally isolated by Thomas D. Brock. It is often abbreviated to

"

Taq

 Pol"  and is frequently used in polymerase chain reaction(PCR), a

method for greatly amplifying short segments of DNA 

.

T. aquaticus

 is a bacterium that lives in hot springs and hydrothermal

vents,  and 

Taq

 polymerase was identified as an enzyme able to

withstand the protein-denaturing conditions (high temperature)

required during PCR. Therefore it replaced the DNA polymerase from 

E.

coli

 originally used in PCR. 

Taq'

s optimum temperature for activity is 75–

80°C, with a half-life of greater than 2 hours at 92.5°C , and can replicate

a 1000 base pair strand of DNA in less than 10 seconds at 72°C. One

of 

Taq'

s drawbacks  

مساوئ 

is its relatively low replication fidelity  

دقة  

It

lacks a 3

'

 to 5

'

 exonuclease proofreading activity, and has an error rate

measured at about 1 in 9,000 nucleotides. 

 Some thermostable DNA

polymerases have been isolated from other thermophilic bacteria and

archaea, such as  

vent  and Pfu

 DNA polymerase, possessing a

proofreading activity, and are being used instead of (or in combination

with) 

Taq

 for high-fidelity amplification.

Enzymes involve in DNA replication



DNA Helicase  Also 

known as helix destabilizing enzyme cases

formation of 

Replication Fork 

due to broken  hydrogen bonds



DNA Polymerase 

Builds a new duplex DNA strand by adding

nucleotides in the 5' to 3' direction.  performs proof-reading and

error correction.



DNA clamp

: A protein (unit from polymerase which prevents

DNA polymerase III from dissociating from the DNA parent

strand.



Single-Strand Binding (SSB) Proteins  

Bind 

to ssDNA and prevent

the DNA double helix from re-annealing after DNA helicase

unwinds it thus maintaining the strand separation



DNA Gyrase 

(and Topoisomerase IV) ; this is a specific type of

topisomerase II convert relaxed form to super coiled



DNA Ligase 

Re-anneals the semi-conservative strands and

joins 

Okazak’i Fragments 

of the lagging strand.



Primase

 Provides a starting point for DNA polymerase to begin

synthesis of the new DNA strand.



Topoisomerase

  I :  Relaxes the DNA from its super-coiled nature



Telomerase 

Lengthens telomeric DNA by adding repetitive

nucleotide sequences to the ends of 

eukaryotic chromosomes

Some important and basic information



Primase:  in fact is RNA polymerase thus the formed primer

Primase:  in fact is RNA polymerase thus the formed primer

is RNA rather than DNA and it will removed latter by DNA

is RNA rather than DNA and it will removed latter by DNA

polymerase I

polymerase I



Topoisomerase I: will break the 3́́  5́ phosphodiester bond

Topoisomerase I: will break the 3́́  5́ phosphodiester bond

converting super coiled to relax form which opposite to ligase

converting super coiled to relax form which opposite to ligase



Helicase : will break hydrogen bond between the two strand

Helicase : will break hydrogen bond between the two strand



Movement of replication fork 5́ →

Movement of replication fork 5́ →

3́́ which is the same direction of

3́́ which is the same direction of

polymerization and direction of Leading strand (

polymerization and direction of Leading strand (

الشريط القائد

الشريط القائد

)

)

     While the direction of lagging strand(

     While the direction of lagging strand(

الشريط الخامل

الشريط الخامل

) is

) is

 3́́ →5́

 3́́ →5́



Polymerization in leading strand is

Polymerization in leading strand is

 continuously 

 continuously 

but it is 

but it is 

un

un

continuously 

continuously 

in lagging strand thus okazaki fragment will form

in lagging strand thus okazaki fragment will form



Okazaki

Okazaki

 fragments are between 1,000 and

 fragments are between 1,000 and

2,000 nucleotides  long in 

2,000 nucleotides  long in 

Escherichia coli

Escherichia coli

 and are between

 and are between

100 and 200 nucleotides long in eukaryotes. They are

100 and 200 nucleotides long in eukaryotes. They are

separated by ~10-nucleotide RNA primers and are un ligated

separated by ~10-nucleotide RNA primers and are un ligated

until RNA primers are removed, followed by enzyme ligase

until RNA primers are removed, followed by enzyme ligase

connecting (ligating) the two Okazaki fragments into one

connecting (ligating) the two Okazaki fragments into one

continuous newly synthesized complementary strand

continuous newly synthesized complementary strand

Types of DNA polymerase in Prokaryotic cell

Types of DNA polymerase in Eukaryotic cell



The DNA 

polymerases of eukaryotes 

polymerases of eukaryotes 

are in general less

are in general less

understood than the DNA polymerases of prokaryotes,

understood than the DNA polymerases of prokaryotes,

Eukaryotic cells have at least five  major nuclear DNA

Eukaryotic cells have at least five  major nuclear DNA

polymerases: 

polymerases: 

α

α

الفا

الفا

  ,

  ,

β

β

بيتا 

بيتا 

,

,

γ

γ

, كاما 

, كاما 

δ

δ

 , دلتا 

 , دلتا 

ε

ε

كاما .    ابسلون 

كاما .    ابسلون 

is found in

is found in

mitochondria, although it is encoded by a nuclear gene. Plant

mitochondria, although it is encoded by a nuclear gene. Plant

chloroplasts also contain their own DNA polymerase that appears

chloroplasts also contain their own DNA polymerase that appears

to be similar to 

to be similar to 

γ

γ



 Pol

 Pol

 α

 α

  polymerase

  polymerase

: it is the only enzyme has  primase activity

: it is the only enzyme has  primase activity

beside DNA polymerase so it is self- primed it will form short

beside DNA polymerase so it is self- primed it will form short

primer 12-20 nts called the initiator RNA  iRNA)

primer 12-20 nts called the initiator RNA  iRNA)



Pol

Pol

 β

 β

 polymerase

 polymerase

: excision repair and it is not highly active and is

: excision repair and it is not highly active and is

not very processive.   

not very processive.   



Pol

Pol

 γ

 γ

  polymerase

  polymerase

: polymerization the mitochondrial DNA  beside

: polymerization the mitochondrial DNA  beside

repairing by its exonuclease activity  3́→5́

repairing by its exonuclease activity  3́→5́



Pol

Pol

δ

δ

 دلتا  

 دلتا  

and 

and 

ε

ε

 ابسلون 

 ابسلون 

polymerase 

polymerase 

:polymerization lagging (

:polymerization lagging (

δ

δ

)and leading

)and leading

(

(

ε

ε

) strand respectively 5 ́→3́.

) strand respectively 5 ́→3́.

 In

 In

 eukaryotes

 eukaryotes

, the low-processivity

, the low-processivity

initiating enzyme, Pol α, has intrinsic primase activity. The high-

initiating enzyme, Pol α, has intrinsic primase activity. The high-

processivity extension enzymes are Pol δ and Pol ε

processivity extension enzymes are Pol δ and Pol ε

.

.

Differences between leading and lagging strand replication

A new DNA strand is always synthesized in a 5’ to 3’ manner, thus

the replication of both the strands goes in  two different ways.



1-  Leading strand                              .

A leading strand is the strand which is run  from  5’-3’direction

or the direction the same as the replication fork movement. It is

synthesized continuously; there are no breaks in-between. This

strand is formed as nucleotides are continuously added to the 3’

end of the strand after polymerase reads the original DNA

template . Only one primer will require here .no Okazaki

fragment will formed.



 2 DNA polymerase 

molecules are required for  polymerization

the two strands which run together in the same machine

binding together  but still the replication happened in opposite

direction The polymerase involved in leading strand synthesis

is DNA polymerase III(DNA Pol III) in prokaryotes and

presumably Pol ε

in yeasts. In human cells the 

leading

 and

lagging

 strands are synthesized by 

Pol ε

ابسلون 

and 

Pol δ

,

respectively, within the nucleus and Pol γ in the mitochondria.

[

2- lagging strand:                       

.

A lagging strand is the strand which is synthesized in the 3’-

5’ direction or 

opposite direction 

as to the movement of the

replication fork. It grows or is synthesized away from the

fork. Its movement in the opposite direction is the cause

why it is discontinuous; it is synthesized in fragments. The

primase, which is responsible for adding an RNA primer, has

to wait for the fork to open before putting the primer. The

lagging strands have fragments of DNA which are called

Okazaki fragments.

More than one primer will be necessary here and it will be

removed latter by the exonuclease activity of 

DNA

polymerase (I) which will 

fill the gap between two adjacent

Okazaki fragments .the final binding will done by the

activity of ligase enzyme  who will add a 3́

 5́

 phospho diester

bond continuously ; this is the reason why the synthesis of

the lagging strand is more complicated than the leading

strand.

The DNA replication machinery 

ماكنة تضاعف الدنا



The 

Replisome 

Replisome 

is composed of the following:

is composed of the following:



2 

2 

DNA Pol III enzymes

DNA Pol III enzymes

molecules 

molecules 

, each has a core subunits composed from 3

, each has a core subunits composed from 3

sub units  

sub units  

α

α

, 

, 

ε

ε

 and 

 and 

θ

θ

 subunits.

 subunits.



the α subunit  has the polymerase activity.

the α subunit  has the polymerase activity.



the ε subunit  has 3'

the ε subunit  has 3'

→

→

5' exonuclease activity.

5' exonuclease activity.



the θ subunit  stimulates the ε subunit's proofreading.

the θ subunit  stimulates the ε subunit's proofreading.



2 

2 

β

β

 units  which act as sliding  clamps(

 units  which act as sliding  clamps(

مثبت المتزحلق

مثبت المتزحلق

(

(

keeping  the polymerase

keeping  the polymerase

bound to the DNA  template .

bound to the DNA  template .



2 

2 

τ

τ

 units which acts to dimerism two of the 

 units which acts to dimerism two of the 

core enzymes (α, ε, and θ

core enzymes (α, ε, and θ

subunits).

subunits).



The 

The 

gamma  

gamma  

γ complex 

γ complex 

  which acts as a clamp loader (

  which acts as a clamp loader (

مثبت الحامل او المقود

مثبت الحامل او المقود

)

)

for the lagging strand helping the two β subunits to form one  unit and bind to

for the lagging strand helping the two β subunits to form one  unit and bind to

DNA. The 

DNA. The 

γ 

γ 

unit is made up of 5 subunits which include  

unit is made up of 5 subunits which include  

3  γ 

3  γ 

subunits

subunits

, 1 δ

, 1 δ

subunit , and 1 δ' subunit . The δ is involved in copying of the lagging strand.

subunit , and 1 δ' subunit . The δ is involved in copying of the lagging strand.

Beside  that there are 

Beside  that there are 

Χ

Χ

  and 

  and 

Ψ

Ψ

  which complete the  complex and bind to γ

  which complete the  complex and bind to γ



DNA polymerase III synthesizes base pairs at a rate of around 1000 nucleotides

DNA polymerase III synthesizes base pairs at a rate of around 1000 nucleotides

per second. DNA Pol III activity begins after strand separation at the origin of

per second. DNA Pol III activity begins after strand separation at the origin of

replication. Because DNA synthesis cannot start replication 

replication. Because DNA synthesis cannot start replication 

 , 

 , 

an 

an 

RNA primer

RNA primer

,

,

complementary to part of the single-stranded DNA, is synthesized

complementary to part of the single-stranded DNA, is synthesized

by

by

 primase

 primase

 (an

 (an

 RNA polymerase

 RNA polymerase

)

)

Structure and sub units  of

2 DNA polymerase III(replisome)

Steps of DNA replication



1- Initiation:  the process require 

replictor

 and 

intiator 

protein(DnaA protein )

. 

For a 

cell to divide

, it must first

replicate its DNA.This process is initiated at particular points in

the DNA, known as replicator (200-300 bp)  which contain

specific area called "

origin of replication ori c  or Dna A box) 

",

which will  opened by  

initiator proteins

. In

E. coli

 this protein is

called  

DnaA protein  

; in

 yeast

, is called  

origin recognition

complex. 

 Sequences opened  by initiator proteins tend to be "AT-

rich" (rich in adenine and thymine bases), because A-T base pairs

have two hydrogen bonds (rather than the three  bond in a C-G

pair). Once the origin has been recognized  ,the initiators

proteins (

DnaA protein )

start  forming  the 

pre-replication

complex

, which unwind  the double-stranded DNA .All known

DNA replication systems require a free 3'

 hydroxyl 

group before

synthesis can be initiated . use a

 primase enzyme

(RNApolymerase)

 to synthesize a short RNA primer(10-20 bp) with a

free 3′ OH group which is subsequently elongated by a DNA

polymerase in this mechanism,



In eukaryotes, primase is produce  by Pol α DNA polymerase  and 

Pol

δ/Pol ε

  are responsible for extension of the primed segments

:

Replication fork 

The replication fork is a structure that forms

during DNA replication .Many enzymes are involved in the

DNA replication fork in order to stabilize initiation step  .

 helicases, which break the hydrogen bonds holding the two DNA

strands together. The resulting structure has two branching

"prongs", each one made up of a single strand of DNA. These

two strands serve as the template for the leading and lagging

strands, which will be created as DNA polymerase matches

complementary nucleotides to the templates; the templates

may be properly referred to as the leading strand template and

the lagging strand templates. SSBPs  also required here

2- Elongation step

DNA is always synthesized in the 5' to 3' direction.

 Since the

leading and lagging strand templates are oriented in opposite

directions at the replication fork, a major issue is how to

achieve synthesis of nascent (new) lagging strand DNA, whose

direction of synthesis is opposite to the direction of the growing

replication fork.

 1-The leading strand receives one RNA primer while the lagging

strand receives several

2-  The leading strand is continuously extended from the primer

by a high 

processivity (

متنامي 

,

متقدم وعامل 

)

, replicative DNA

polymerase, while the lagging strand is extended

discontinuously from each primer, forming Okazaki fragments

As DNA synthesis continues, the original DNA strands

continue to unwind on each side of the bubble, forming

a replication fork

 with two prongs(

شوكة

)

3-

β

Clamp proteins : it 

 form a sliding clamp around DNA,

helping the DNA polymerase maintain contact with its

template, thereby assisting with processivity. The inner face of

the clamp enables DNA to be threaded through it. Once the

polymerase reaches the end of the template or detects double-

stranded DNA, the sliding clamp undergoes a conformational

change that releases the DNA polymerase. Clamp-loading

proteins are used to initially load the clamp, recognizing the

junction between template and RNA primers

.



Lagging strand :it 

 is synthesized in short, separated

segments. On the lagging strand 

template

,

a primase "reads" the template DNA and initiates

synthesis of a short complementary RNA primer. A DNA

polymerase III  extends the primed segments,

forming Okazaki fragments.  DNA polymerase  will add

nucleotides in the 5' to 3' direction; however, one of the

parent strands(lagging) of DNA is 3' to 5' while the other

(leading) is 5' to 3'. To solve this problem, replication

occurs in opposite directions. lagging strand run  away

from the replication fork, and synthesized a series of

short fragments known as Okazaki fragments,

consequently requiring many primers. The RNA primers

of Okazaki fragments are  degraded by Rnase H and DNA

Polymerase I

3-Termination



1-Termination requires that the progress of the DNA replication fork must stop

or be blocked. Termination at a specific locus, when it occurs, involves the

interaction between two components: (1) a termination site sequence in the

DNA, and (2) a protein which binds to this sequence to physically stop DNA

replication. In various bacterial species, this is named the DNA replication

terminus site-binding protein, or 

Ter protein

.



2-Because bacteria have circular chromosomes, termination of replication

occurs when the two replication forks meet each other on the opposite end of

the parental chromosome . As a result, the replication forks are constrained to

always meet within the termination region of the chromosome.

3-Removes the primer (RNA fragments), by  

DNA polymerase I 

by 5'-3'

exonuclease activity of polymerase I, and replaces the RNA nucleotides with

DNA nucleotides.  and   fill the gaps.

4-  When this is complete, a single nick on the leading strand and several nicks

on the lagging strand can be found.

5- 

Ligase  

works to fill these nicks in, thus completing the newly replicated DNA

molecule  .

6- 

Topoisomerase IV 

will : separate the two complete daughter  chromosome in

to two chromosome

Termination in Eukaryotic cell



Primer removal in eukaryotes is  performed by  

RNase I 

that

remove all the primer leaving only one nucleotide in the

junction between 2 nucleotide and the remained one will

removed by 

FenI enzyme . 

Eukaryote cell initiate DNA

replication at multiple points in the chromosome, so

replication forks meet and terminate at many points in the

chromosome; these are not known to be regulated in any

particular way. Because eukaryotes have linear chromosomes,

DNA replication is unable to reach the very end of the

chromosomes, but ends at the 

Telomere 

region of repetitive

DNA close to the end. This shortens the telomere of the

daughter DNA strand. Shortening of the telomeres is a normal

process in 

Somatic

cells. 

As a result, cells can only divide a

certain number of times before the DNA loss prevents further

division. Within the 

Germ cell

 line, which passes DNA to the

next generation, 

Telomerase

 extends the repetitive sequences

of the telomere region to prevent degradation. Telomerase can

become mistakenly active in somatic cells, sometimes leading

to 

Cancer 

formation. in the end of the replication the DNA will

warp  around the basic histons to form the chromatin