Lecture notes on Prokaryotic DNA Replication by Himani Gandhi, Assistant Professor in Microbiology, RK University, Rajkot.

1. Miss.Himani Gandhi.
AssistantProfessor,RK University,Rajkot
Molecular Basis of Microbial Genetics
REPLICATION OF DNA
DNA Replication
“DNA replication is the biologicalprocess of producing two identical copies of DNA
from one original DNA molecule”
“DNA can make exact copies of itself, this process is known as replication”
Replication occurs in all living organisms and it is basis for biological inheritance.
Single stranded DNA is first converted to a double stranded molecule; which then serves
as a template for synthesis of complementary ss-DNA.
The primary role of any mode of replication is to the duplicate the base sequence of the
parent molecule.
The specificity of base pairing adenine (A) with thymine (T) and guanine (G) with
cytosine (C).
The result of replication is the formation of two double helices; one strand is daughter
and second is parental strand.
The two strands of a DNA are combined by hydrogen bonds between the purine (A & G)
and pyrimidine (T & C) base pairs.
In a cell, DNA replication starts at specific location that knows as origins of the genome
which is rich in adenine and thymine.
A number of proteins and enzymes are associated with the replication fork which helps
in initiation, continuation and termination of DNA synthesis.

2. Miss.Himani Gandhi.
AssistantProfessor,RK University,Rajkot
Molecular Basis of Microbial Genetics
REPLICATION OF DNA
Meselson - Stahl Experiment
Semi conservative Mode of DNA Replication
Delbrucksuggestedthatthe Watson - Crick Model of DNA couldtheoreticallyreplicate bythree
modes;suchas conservative,dispersiveandsemi conservative.
1. According to the conservative mode of replication, the two double helices of DNA formed in
which one daughter molecule contains both DNA stands and the other daughter molecule
contains newly synthesized DNA stands. Thus, the old parental double helix would be
unchanged.
2. According to the semi conservative mode of replication, the two parental DNA strands
separate and each strands acts as a template for the synthesis of a new DNA strands. Thus
result is two double helix formed that consists one parental DNA strands and one new DNA
strands.
3. According to the dispersive mode of replication, the parental double helix of DNA is broken into
double stranded segments that, acts as templates for the synthesis of new double helix
molecule.
Figure 1. Mode of DNA Replication

3. Miss.Himani Gandhi.
AssistantProfessor,RK University,Rajkot
Molecular Basis of Microbial Genetics
REPLICATION OF DNA
In 1958, Mathew Meselson and Franklin Stahl demonstrated the semi conservative nature of
replication.
E. coli grownfor several generationsinamediumcontaining“heavynitrogenisotope”(N15).
All the nitrogen of the bacteria including that of DNA became N15; which is heavier than ordinary
DNA
The densityof the DNA distinguishedbyultracentrifugationusingacesiumchloride (CsCl) gradient.
Now, cells with N15 labeled DNA grown in “light nitrogen N14” containing media, an allowed to divide
several times.
After the first division the DNA was extracted that was found to be a hybrid as a (N14 + N15). This
hybridwasnot as heavyas N15 noras lightas N14, buthadan intermediate density.
After the second division two kinds of DNA were found, normal N15 DNA (50%) and hybrid N14 + N15
DNA (50%).
Afterthe thirddivision(75%) ¾of the DNA wasnormal N14 DNA and (25%) ¼ washybridN14 + N15.
After experiment; result shows that pattern of extracted DNA could only have been observed, if DNA
molecules contains a template strand from the parental DNA; thus DNA replication is semi
conservative.
Figure 2. Meselson and Stahl Experiment for Semi conservative mode of replication

4. Miss.Himani Gandhi.
AssistantProfessor,RK University,Rajkot
Molecular Basis of Microbial Genetics
REPLICATION OF DNA
Protein Machinery of DNA Replication
Enzymes involves in DNA Replication
The followingproteinsorenzymesare involvedinDNA replication.
A. ProteinsinvolvedinInitiationof DNA replicationor involvedinopeningofDNA helix.
1. Helicase/DnaBprotein
2. Single StrandedBindingProtein(SSBproteins)
3. DNA Topoisomerase/DNA Gyrase
B. Proteinsor enzymesinvolvedinElongation of DNA replication
4. DNA Primase (DnaG)
5. DNA Polymerase III(DNA pol III)
6. DNA Polymerase I
7. DNA Ligase
C. ProteinsinvolvedinTermination ofDNA replication
8. Tus Proteins
Figure 3. DNA Replication Fork

5. Miss.Himani Gandhi.
AssistantProfessor,RK University,Rajkot
Molecular Basis of Microbial Genetics
REPLICATION OF DNA
A. Proteins involved in Initiation of DNA replication or involved in opening of
DNA helix.
There are three types of proteins that are Helicase, SSB proteins and DNA Topoisomerase
required for to open DNA double helix and to provide single stranded DNA as a template for DNA
replication
1. Helicase/ Rep proteins/Dna B protein:
DNA helicase orRepproteinsare knownas unwinding proteins.
During DNA replication, the ds-DNA must be separated into ss-DNA. This ss-DNA will be used as
a template forreplication.
DNA helicase/ Rep protein hydrolyzed ATP, when they bind with ss-DNA and move along with
DNA helix by breaking the hydrogen bonds between the ds-DNA and separate the strands to
formreplicationforkwhichcomparable withazipopener.
DNA helicase ishexamericprotein
The hydrolysis of ATP changes the shape of a helicase that allow the helicase to perform
mechanical work.
2. Single Stranded Binding Protein/SSB Protein:
The SSB proteins bind with ss-DNA which kept the two strands separate and provide the
template fornewDNA synthesis.
It protectsthe degradationof ss-DNA bynucleases.
3. DNA Topoisomerase/DNA Gyrase:
The supercoils are formed at the other side because the double helix of DNA separates from one
side due toaction of helicase;thenreplicationproceeds.
The problem of supercoils are solved by a group of enzymes called DNA Topoisomerase that also
knownas DNA gyrase.
It break a phosphoidester bond in either ss-DNA or ds-DNA to solved supercoils, so DNA
Topoisomerase also known as “reversible nuclease”. This process does not require additional
energy.
There are twotypesof DNA Topoisomerase.
Type I DNA Topoisomerase:
It cuts the ss-DNA toovercome the problemof supercoils.
Theyhave bothnuclease (strandcutting) andligase (strandsealing) activity.
Theydo not require ATP.
Type II DNA Topoisomerase:
It binds tightly to DNA double helix and cuts both strand (ds-DNA) and reseal them
to overcome the problemof supercoils.
TheyusedenergyfromATPhydrolysis.

6. Miss.Himani Gandhi.
AssistantProfessor,RK University,Rajkot
Molecular Basis of Microbial Genetics
REPLICATION OF DNA
Figure 4. Role of DNA gyrase to solve supercoils
B. Proteins involved in Elongation of DNA replication
There are four proteins and enzymes which are DNA Primase, DNA polymerase III, DNA
polymerase IandDNA ligase involvedinelongationof DNA replication.
4. DNA Primase/Dna G:
Initiation of DNA synthesis requires a short polynucleotide with a 5’ triphosphate end & a 3’-OH
endthat knownas RNA primers.
Thishydroxyl groupservesasthe firstacceptorof nucleotidebyactionof DNA polymerase.
RNA primerssynthesizedbyDNA Primase byusingthe DNA template.
DNA polymerase cannotinitiateDNA synthesis.
The enzyme primase associated with SSB proteins forms a complex called primosomes that
produce RNA primers.
Primase synthesizes the RNA primers; approximately ten nucleotide long that are
complementaryandantiparallel toDNA template.
As a result of primosomes; RNA primers are constantly being synthesized at replication fork on
laggingstrand.
Onlyone RNA sequence issynthesizedatoriginof replicationonthe leadingstrand.

7. Miss.Himani Gandhi.
AssistantProfessor,RK University,Rajkot
Molecular Basis of Microbial Genetics
REPLICATION OF DNA
Figure 5. Role of RNA primers in DNA synthesis.
5. DNA Polymerase III (DNA pol III):
DNA polymerase playsanessential role inDNA replication.
It is multimeric enzyme or holoenzyme having ten subunits such as alpha (α), beta (β), epsilon
(ε),theta(θ),tau (τ),gamma (γ),delta(δ),deltadash(δ’),chi (χ),andpsi (ψ).
All these tensubunitsare neededforDNA replicationandall havingdifferentfunctions.
The holo enzymes made up of all ten units; while the core enzyme made up of the three
subunitsalpha(α),beta(β),andtheta(θ).
Remainingsevensubunitsincreaseprocessivityof DNA replication.
There are twofunctionof DNA pol III inDNA replication.
1. 5’----->3’Polymerizationactivity(Chainelongation)
The synthesis of a new DNA strand catalyzed by DNA pol III in 5’ -----> 3’ direction,
whichisantiparallel tothe parentDNA strand.
It is required four deoxyribonucleoside triphosphate dATP, dGTP, dCTP, and dTTP
(dNTPs).
2. 3’----->5’ Exonuclease activity(Proof readingactivity)
Fidelity of replicationismostimportantforthe organisms.
DNA pol III checks the incoming nucleotides on growing DNA strand and removes
mispairsof nucleotidebases,thisprocessknownas‘proof readingactivity’.
The exonuclease activity catalyzed in 3’-----> 5’ direction to remove the mismatched
nucleotide.
6. DNA Polymerase I (DNA pol I)/Kornberg Enzyme:
It isdiscoveredbyArthurKornberg;soitalso knownasKornbergEnzyme.
There are three functionof DNA pol Iin DNA replication.
1. 5’----->3’Polymerizationactivity
DNA pol I have polymerizationactivity in5’----->3’ direction&fill the gaps.
It isDNA dependentactivityandrequiringa3’ primersite anda template strand.

8. Miss.Himani Gandhi.
AssistantProfessor,RK University,Rajkot
Molecular Basis of Microbial Genetics
REPLICATION OF DNA
2. 3’----->5’ Exonuclease activity
DNA pol I act as 3’-----> 5’ exonuclease which proofreads the newly synthesized
DNA.
If polymerase I erroneously incorporates a wrong nucleotide at the end of growing
DNA chain; polymerization activity is inhibited and 3’-----> 5’ exonuclease remove
the offendingnucleotide.
3. 5’----->3’ Exonuclease activity
RNA primersare removedandreplacedatthe endsof the newlysynthesizedDNA.
DNA pol I act as 5’-----> 3’ exonuclease that is hydrolytically remove the RNA
primers.
It also remove pyrimidine dimers (T=T); which are formed when DNA is exposed to
ultravioletlight.
Figure 6. Removal of RNA primer and fillingofthe resultinggaps by DNA pol I
7. DNA ligase:
DNA ligase catalyzed the formation of a phosphoidester linkage between the 5’ phosphate
group on the DNA which synthesized by DNA pol III and the 3’ hydroxyl group on the DNA
(Okazaki pieces)whichmade byDNA pol I;thisprocessknownas ‘nick-sealing’.
It require the energythatutilizedbythe breakdownof ATPtoAMP & Ppi.

9. Miss.Himani Gandhi.
AssistantProfessor,RK University,Rajkot
Molecular Basis of Microbial Genetics
REPLICATION OF DNA
C. Proteins involved in Termination of DNA replication or stop the DNA
replication.
There are Ter sitesandTus proteinsinvolvedinterminationof DNA replication.
8. Tus proteins:
The E.coli replication terminus is a large region; where sevenidentical non-palindromic (~ 23 bp)
terminates sites such as TerE, TerD and TerA on the one side and TerG, TerF, TerB and TerC on
the otherside that isterminate the replicationof DNA.
A replication fork travelling counter clockwise passes through TerG, TerF, TerB and TerC; but
stopson TerA, TerD and TerE.
Similarly a clockwise travelling replication fork transits TerE, TerD and TerA; but halt at TerC or
TerB or TerF or TerG.
Ter sites arrest the movement of replication fork by binding of Tus protein which is a product of
tus gene.
Tus proteins bind specifically to Ter sites and inhibit the function of DNA helicase. Thereby
replicationisterminated.
Figure 7. The positions of the Ter sites and the OriC site

10. Miss.Himani Gandhi.
AssistantProfessor,RK University,Rajkot
Molecular Basis of Microbial Genetics
REPLICATION OF DNA
DNA Replication (Y shaped model of DNA Replication)
Steps involves in DNA Replication
“DNA replication is the biologicalprocess of producing two identical copies of DNA
from one original DNA molecule”
DNA replication is completed in following three steps:
1. Initiation
2. Elongation
3. Termination
1. Initiation:
Ori C site/ Origin:
The DNA replication always begins by separating a twisted strand into two
untwistedmolecularstrands.
It happensinthe specificareaof a strand knownasthe “origins".
The origins contain a single unique nucleotide sequence approximately 245 bp that
knownas origin.
It isrich inadenine andthymine (A &T).
It is utilized AT rich region as initiator because A-T base pair have two hydrogen
bondsratherthan in G-Cbase pair have three bonds,whichare easilytobreak.
Helicase:
Ori C region codes attract the helicase, a protein that responsible for the separation
of the strands.
Once the helicase locates the origins, it sends out signals inside the cell for other
replicationinitiatorproteinstohelpoutinthe separation.
DNA strands are being separated by breaking the hydrogen bonds between the
nucleotide base pairs, such as those of adenine (A) and thymine (T), and guanine (G)
and cytosine (C).
Resultof helicase replicationforksisformed; whichare extendedbidirectionally.
SSB proteins:
A single strand binding protein prevents rewinding of the DNA and protects the
degradationof single strandedDNA bynuclease.

11. Miss.Himani Gandhi.
AssistantProfessor,RK University,Rajkot
Molecular Basis of Microbial Genetics
REPLICATION OF DNA
Figure 8. DNA replication

12. Miss.Himani Gandhi.
AssistantProfessor,RK University,Rajkot
Molecular Basis of Microbial Genetics
REPLICATION OF DNA
DNA Topoisomerase:
Topoisomerase IandII preventsupercoiling.
Due to action of helicase andSSBproteins,supercoilsare formed.
Topoisomerase breakthe bondsineitherss-DNA ords-DNA tosolvedsupercoils.
2. Elongation:
DNA primase:
It synthesizes shortRNA primerswhichare complementarytothe DNA strand.
DNA pol III:
DNA Pol III starts adding nucleotide in 5’----> 3’ direction to the 3’ -OH end of the
primersthatis the polymerizationactivityof pol III.
It has alsoproofreadingactivityinwhichitreplacedmismatchedbase paring.
DNA pol III synthesizes new DNA forbothleadingandlaggingstrand.
Leadingstrand:
On the template strand with 3’----> 5’ orientation, new DNA is synthesized
continuouslyin5’---->3’directiontowardsthe replicationfork.
The new strand that is continuously synthesized in 5’----> 3’ direction is the leading
strand.
Lagging strand and Okazaki fragments:
On the template strand with 5’----> 3’ orientation, multiple primers are synthesized
at specific sites by primase (primosomes) and DNA pol III synthesize short pieces of
newDNA (about1000 nucleotideslong) in5’---->3’direction.
These small DNA fragments are discontinuously synthesized are called Okazaki
fragmentsdiscoveredbyReigi Okazaki.
The new strand which is discontinuously synthesized in small fragments is called the
laggingstrand.
DNA pol I:
After DNA synthesis by DNA pol III, DNA pol I uses its 5’----> 3’ exonuclease activity
to remove the RNA primersandfillsthe gapsbyaddingdNTPsinnew DNA strand.
DNA ligase:
The gap between the two DNA fragments (Okazaki pieces) is sealed by DNA ligase
and create continuousnew DNA strand.

13. Miss.Himani Gandhi.
AssistantProfessor,RK University,Rajkot
Molecular Basis of Microbial Genetics
REPLICATION OF DNA
3. Termination:
Tus proteins/Ter sites:
The replication fork moves ~ 180 degree opposite to Ori C because DNA is circular
inprokaryotes.
Around this region there are seven terminator sites which arrest the movement of
replication fork by binding to the tus gene product (Ter sites), and inhibit the
functionof helicase.
DNA Topoisomerase II:
Once replication is completed, the two double stranded circular DNA molecules
(daughter strand) remain interlinked. So Topoisomerase II cut ds-DNA to unlink
and releasedtwodaughtersDNA.

14. Miss.Himani Gandhi.
AssistantProfessor,RK University,Rajkot
Molecular Basis of Microbial Genetics
REPLICATION OF DNA
Model of DNA Replication
The bacterial chromosome consists of a double stranded DNA ring attached to the plasma
membrane. Usually there is a single replication fork during replication. This starts at a point called origin
and moves around the chromosome. During rapid growth there may be one or two additional forks
formed at the origin before the first one completes its replication cycle. Several models have been
proposedtoexplainthe mechanismof replicationincircularchromosomes.
Here, there are three models explain the mechanism of DNA replication in circular such are the
Cairnsmodel,the Yoshikawamodel andthe rollingcircle model.
1. The Cairns Model (1963):
The cairns model of replication have been demonstrated in the bacteria E.coli and
Bacillus subtilis, in several viral and plasmid chromosomes and in DNA synthesis of
mitochondriaandchloroplasts.
Figure 9. Cairns Model for circular DNA replication.
A) Double helix DNA.
B) The ds-DNA denaturedatoriginsite.DNA fragmentinitiated denovo.
C) A secondfragmentinitiatedandasecondgrowingpointestablished.
D) As a result of bidirectional replication the two growing points move forward. This
causes super twisting of unreplicated portion of DNA resulting in conformational
strainon the molecule.

15. Miss.Himani Gandhi.
AssistantProfessor,RK University,Rajkot
Molecular Basis of Microbial Genetics
REPLICATION OF DNA
E) Swiveling protein relieves the strain by causing ss-nicks, thus allowing free rotation
of the parental strands with reference to each other. The swiveling protein then
sealsthe nicks.
F) Replicationproceedsandthe twogrowingpointsmeetonthe terminus.
2. The YoshikawaModel:
A variationof the Cairnsmodel hasbeensuggestedbyYoshikawa.
Accordingto thismodel the newlyformedDNA strandsbecome covalentlyjoinedtothe
endsof the parental chromosomes.
Figure 10. Yoshikawa’s model of replication
3. The Rollingcircle Model:
Gilbert and Dressler, Eisen, Pereira da Silva and Jacob in 1968 explain rolling circle
model of replication in single stranded DNA viruses (θX174) and the transfer of E.coli sex
factor (Plasmid)
A) Single strandedDNA (ss-DNA) ringof θX174 that ispositive (+).
B) Synthesis of negative strand (-) and formation of double stranded (ds-DNA)
replicative form.
C) Nickingof one parental strandbyendonuclease.
D) Parental strands rolls and unwinds. 5’ end attached to the host membrane. New
DNA synthesizedonthe 3’endand at the “tail”.

16. Miss.Himani Gandhi.
AssistantProfessor,RK University,Rajkot
Molecular Basis of Microbial Genetics
REPLICATION OF DNA
E) Further unwinding and synthesis carried out. The tail region synthesizes
discontinuoussegments.
F) Tail is cut bya specificendonuclease intounitlengthprogenyrod.
G) Circularization of rods to form new circular molecules which can become new
rollingcircles.Duringcircularizationthe gapisclosedbya ligase.
Figure 11. Rolling circle model of DNA replication.

17. Miss.Himani Gandhi.
AssistantProfessor,RK University,Rajkot
Molecular Basis of Microbial Genetics
REPLICATION OF DNA
Summary of DNA Replication
DNA can make exactcopiesof itself,thisprocessknownasDNA Replication.
It isbasisfor biological inheritance.
DNA replicationissemi conservativeprocesswhich explainedbyMeselsonandStahl.
It takesplace duringthe interphase.
DNA replicationcarriedoutinthree stepssuchas,Initiation,ElongationandTermination.
DNA unwindsatthe originof replication.
Helicase opensupthe DNA formingreplicationforks;theseare extendedbidirectionally.
Single strand binding proteins coat the DNA around the replication fork to prevent
rewindingof the DNA.
Topoisomerase bindsatthe regionaheadof the replicationforktopreventsupercoiling.
Primase synthesizesRNA primerscomplementarytothe DNA strand.
DNA polymerase startsaddingnucleotidestothe 3'OH endof the primer.
Elongationof boththe laggingandthe leadingstrandcontinues.
RNA primersare removedbyexonuclease activity.
Gaps are filledbyDNA pol byaddingdNTPs.
The gap betweenthe twoDNA fragmentsissealedbyDNA ligase,whichhelpsinthe
formationof phosphoidesterbonds.
ReplicationterminatedatTer sites.
There are three modelsforcircularreplicationsuchasCairnsModel,Yoshikawamodel and
Rollingcircle model.