A comprehensive presentation on Prokaryotic and Eukaryotic DNA Replication with their clinical applications for MBBS , BDS, B Pharm & Biotechnology students to facilitate self- study.

1. Prokaryotic and Eukaryotic DNA Replication with
their clinical applications
Dr. Rohini C Sane

3. Identical
base sequences
5’
5’
3’
3’ 5’
5’
3’
3’
WatsonandCrickdouble-strandedDNAstructureTwisted Ladder (B form and
double helix)
1. Right handed helix
(two deoxy
ribonucleotide
strands around each
other )
2. Anti- parallel strands :
(5’→3’ &3’→5’ )
3. Width (diameter ):
20A (2 nm )
4. Each turn of pitch =
34 nm (3.4nm )with
10 base pairs at the
distant of 3.4nm
5. Hydrophobic bases
stacked inside &
hydrophilic
deoxyribose
nucleotides periphery
6. Stands are identical
but complementary
due to base pairing

4. Hydrogen bonds holding two strands of DNA Structure
Watson & Crick Model of DNA Structure :Two stands are held together by hydrogen bonds,
Hydrogen bonds between Purine & Pyrimidine ,A= T ,C  G (CG Pair stronger than at pair)

5. Major and minor grooves in DNA structure
Each turn of pitch = 34 nm (3.4nm )with 10 base pairs at the distant of 3.4nm

6. DNA as a long term repository of genetic information
1. DNA is a bank of genetic information & controls protein biosynthesis.
2. Single mammalian cell contains 10 picograms (10-12 gm )of DNA.
3. DNA (long term repository of genetic information) is more stable than
RNA.
4. DNA in cell must be duplicated ,maintained & accurately pass down to
the daughter cell.

7. Functions of Deoxy-ribonucleic Acids(DNA)
❖Functions of Deoxy-ribonucleic Acids(DNA):
1. Chemical basis of hereditary & reserve bank of genetic information
2. Maintain identity of different species over million years
3. Control cellular functions
4. DNA possess organized genes which control protein synthesis
5. DNA→RNA→PROTEIN ( central dogma)
6. Mitochondrial DNA → specified function with respect to protein
synthesis

8. Organization of Prokaryotic and Eukaryotic DNA
• Organization of Prokaryotic DNA: as a single chromosome in in the form of
double-stranded circle. It is packed in the form of nucleotides by interaction
with proteins and certain cations( polyamines ).
• Organization of Eukaryotic DNA: DNA is associated with Histone proteins to
form chromatin which then gets organized into compact structures namely
chromosomes.
• Nucleosomes : two turns of DNA(150bp)wrapped around core(Histone
proteins H1,H2B,H3,H4 two molecules each) .

9. Packing of DNA into chromosomes:
1. Level 1 Winding of DNA around histones to create a nucleosome
structure.
2. Level 2 Nucleosomes connected by
strands of linker DNA like
beads on a string.
3. Level 3 Packaging of nucleosomes into
30-nm chromatin fiber.
4. Level 4 Formation of looped domains.
Organization of Eukaryotic DNA
30 nm fibers organized into
loops by anchoring the fiber
at A/T rich regions (scaffold
–associated regions SARS )
to protein scaffold.
During mitosis, the loops
are further coiled , the
chromosomes condense
and become visible.


10. The central dogma of molecular biology:1
❑The central dogma of molecular biology states that genetic information
flows DNA to RNA to Protein by processes of
1. DNA Replication :that involves formation of daughter DNA molecules
using a parental DNA as a template .
2. Transcription of DNA: in which genetic information in DNA is
transcribed to form messenger RNA(m- RNA).
3. Translation of m-RNA into amino acid sequence of protein.
❖DNA Replication →Transcription of DNA →Synthesis of m-RNA
→Translation of m-RNA →Protein Synthesis
❖In retroviruses which have RNA genomes, genetic information flows in
reverse direction i.e. from RNA to DNA.

11. The central dogma of molecular biology
DNA
RNA
Protein
Transcription
Translation
Reverse Transcription
Replication
The flow of genetic information

12. The central dogma of molecular biology:2

14. Basic requirements for DNA replication
➢The basic requirements and components of replication are the same for
prokaryotes and eukaryotes.
➢Replication in prokaryotes is much better understood than in eukaryotes.
• Substrates: 4 deoxyribonucleosides triphosphate( dATP , dGTP ,dCTP, dTTP)
• Template : separated strands of DNA serve as template for the synthesis of
the new daughter DNA strands .It is required to direct the addition of the
appropriate complementary nucleotide to newly synthesized DNA strand.
• Proteins and Enzymes : different types of DNA polymerases and proteins

15. ❖DNA Replication involves three R :
1. Replication
2. Repair
3. Recombination

16. Proteins involved in initiation of DNA replication at E.Coli origin
Protein Molecular
weight
Numberof
subunits
Function
DNA A Protein 52000 1 Recognizes ori sequences, opens DNA duplex
at a specific site in origin
DNA B Protein(Helicase) 300000 6 Unwinds DNA , primosome constituent
DNA C Protein 29000 1 Required for DNA B binding at origin
HU(Histone like protein) 19000 2 DNA binding protein , stimulates initiation
Primase(DNA G Protein) 60000 1 SynthesizesRNAprimers,primosomeconstituent
SSB(singlestrandedbinding
protein)
75600 4 BindssinglestrandedDNAandstabilizestheseparated
DNAandpreventsrenaturationofDNA
DNAtopoisomeraseII (DNAgyrase) 400000 4 releasestorsionalstraingeneratedbyDNAunwinding
DNA polymerase I 103000 1 Filling gaps and excisions of primers
DNA polymerase III 791500 17 new DNA strand elongation
DNA ligase 74000 1 Sealsthesinglestrandnickbetweenthenascentchain
andokazaki fragmentsonlaggingstrand(ligation)

17. Proteins involved in initiation of DNA replication at E.Coli origin
Protein Molecular
weight
Numberof
subunits
Function
RNA polymerase 454000 5 Facilitates DNA A Protein binding activity
DNA methylase 32000 1 Methylate of (5’ ) GATC sequence at ori C
DNA polymerase I Filling gaps and excisions of primers
Ter binding protein Prevents DNA B Protein(Helicase) from further
unwinding of DNA and facilitates the termination of
replication

18. Comparison of Prokaryotic and Eukaryotic DNA polymerases
Prokaryotic DNA
polymerases
Eukaryotic DNA
polymerases
Functions
I (Pol)  Gap filling and synthesis between Okazaki fragments of
lagging strand(5’ →3’ polymerization activity)
II(Poll)  DNA proof reading and DNA repair(3’→5’exonuclease
activity)
 DNA repair (5’→3’ exonuclease activity)
 Mitochondrial DNA synthesis
III(Polll)  Functions at replication fork, catalyzing Leading and
lagging strand synthesis

19. DNA Replication in Prokaryotes
❖When cell divides a daughter cell receives identical copies of genetic
information from a parent cell.
❖Definition of DNA Replication :Replication of DNA is the process in
which DNA copies produce identical daughter molecules of DNA.
1. DNA Replication exhibits high fidelity which is essential for survival
of fetus.
2. DNA Replication is semi- conservative :half of original DNA is
conserved in the daughter DNA .(Meselson & Stahl 1958)
• Newly synthesized DNA has half of the parental DNA & one half of
new DNA.

20. Mechanism of DNA Replication In Prokaryotes
❖Features of DNA Replication in Prokaryotes:
• Semi- continuous, semi–conservative & bi-directional
• Replication proceeds in 5’→3’ direction
• Simultaneously both strands of DNA
• Replication in Leading strand is continuous & forward .
• Replication in Lagging strand is discontinuous & short pieces of DNA (15-250
nucleotides ).Okazaki fragments are produced on Lagging strand .
• DNA Synthesis :bidirectional from point of origin in replication bubble
• Two replication forks move in opposite directions from replication bubble or
replication eye ,which becomes lager and assumes a  shaped structure.
• 3 Stages of replication : initiation ,elongation and termination.

21. DNA Replication in Prokaryotes: produce identical daughter molecules of DNA
DNA replication : Duplication /synthesis of DNA. It is needed to transfer the genetic
information stored in DNA of parent cell to a daughter cell during cell reproduction.
In semi conservative mechanism , each
replicated duplex daughter DNA
molecules contains one parent strand
and one newly synthesized strand.

22. DNA Replication in Prokaryotes: Semiconservative :
half of original DNA is conserved in the daughter DNA .

23. Three models for DNA replication

24. Components of DNA Replication

25. Components of DNA Replication
• Replicon : is the unit of DNA in which individual acts of replication
occurs. Bacterial chromosome contains a single replicon ,eukaryotic
chromosome has a large number of replicons.
• Replication fork: also known as growing point ,at which replication
occurs. Replication may be unidirectional or multidirectional based on
whether one or more replication forks starts from the origin
respectively .
• Origin of Replication : the site at which replication begins .These sites
are generally AT – rich to facilitate unwinding . Proteins and enzymes
required assembled at origins.

26. Replication forks of DNA in prokaryotes and eukaryotes

27. Overview of bacterial DNA replication
Twotopologicallyinterlinkedcircular
chromosomescalledcatenanes
generatedbyreplicationare
separatedbyatypeII
topoisomeraseandsegregatedinto
twodaughtercellsatcelldivision.

28. Directionality of the DNA strands at a replication fork
Leading strand
Lagging strand
Fork movement
A new strand of DNA is always
synthesized in the 5==3 direction,
with the free 3 OH as the point at
which the DNA is elongated
Replication fork

29. Direction of movement of replication fork

30. DNA replication in prokaryotes :Semi- continuous & bi-directional
DNA Replication in Leading strand is continuous & forward.
Replication in Lagging strand is discontinuous & short pieces of DNA (15-250 nucleotides).
Okazaki fragments are produced on Lagging strand .

31. Semi–conservative DNA Replication In Prokaryotes

32. Replication proceeds in 5’→3’ direction
DNA Synthesis :
bidirectional from point of
origin in replication bubble

33. Functions of Helicases in DNA Replication
❑Helicases separate double stranded DNA to single stranded DNA
during replication and the energy derived from ATP hydrolysis .
❑Helicases unwind the DNA duplex just ahead of the replication fork
at a rate of 1000 bp/s. Two DNA Helicases unwind DNA at a
replication fork moving in opposite directions ,one on the leading
strand template and another on the lagging strand template.
❖Functions of Helicases:
1. DNA unwinding occurs during replication. Helicases in conjunction
with topoisomerase relieve torsional stain .
2. It functions in homologous recombination, nucleotide excision
repair , transcription termination and conjugation.

34. DNA helicase unwinds
the DNA duplex
ahead of DNA polymerase
creating single stranded
DNA that can be used
as a template
Functions of Helicases
in DNA Replication

35. Functions of Helicases in
DNA Replication in Prokaryotes:
DNA unwinding during replication.
Helicases in conjunction with
topoisomerase relieve torsional stain .
Role of Helicases in DNA Replication

36. Uses energy from ATP to
unwind the duplex DNA
SSB
SSB SSB
SSB
Functions of Helicases in DNA Replication

37. Functions of single binding proteins(SSB) in DNA Replication
❖Single binding proteins(SSB):also known as DNA helix destabilizing
proteins or Single stranded DNA binding proteins. They have no enzyme
activity.
❖Functions of SSB Proteins during DNA Replication:
1. Keep two strands of DNA separate(separated by helicases).
2. Bind tightly in a co-operative manner to single stranded DNA
(separated strands) and makes it available as a template for DNA
Replication/ synthesis.
3. Stabilize DNA in a single strand state and prevent base pairing.
4. Protect single stranded DNA degradation by nucleases.

38. Role of single binding proteins(SSB) in DNA Replication:1
ssDNA binding proteins bind to the sugar phosphate backbone leaving the bases exposed for
DNA polymerase.

39. Role of single binding proteins(SSB) in DNA Replication:2
ssDNA binding proteins are required to
“iron out” the unwound DNA
Stabilize DNA in a single strand state and prevent base pairing. Protect single stranded DNA degradation
by nucleases.

40. Role of  clamp in Elongation of DNA Replication in Prokaryotes
❖ As the leading strand is being synthesized ,corresponding portion
of Lagging strand is looped through a  clamp enabling coordinate
synthesis of both strands.
❖Both core complex and  clamp dissociate after synthesis of Okazaki
fragments and again associate the next Okazaki fragment .

41. DNA polymerase is
not very processive
(i.e. it falls off the DNA
easily). A “sliding clamp”
is required to keep
DNA polymerase on and
allow duplication of long
stretches of DNA
Role of  clamp in
Elongation of DNA
Replication in
Prokaryotes

42. Role of sliding clamp  -subunits in Elongation of DNA Replication in Prokaryotes
Astheleadingstrandisbeingsynthesized,correspondingportionofLaggingstrandisloopedthrougha
clampenablingcoordinatesynthesisofbothstrands.Bothcorecomplexandclampdissociateaftersynthesis
ofOkazakifragmentsandagainassociatethenextOkazakifragment.

43. A “clamp loader:” complex is required to get the clamp onto the DNA.
Role of clamp loader

44. Role of Helicases , SSB proteins and Topoisomerase in DNA Replication
Topoisomerase type I and type II play important role in DNA Replication , transcription and
recombination.

45. Positive and Negative supercoils of DNA
PositivesupercoilsofDNAareformedwhenthe
DNAmoleculeistwistedinthesamedirectionasthe
righthandedhelixofB-formDNAaboutitsaxis.
NegativesupercoilsofDNAareformedwhentheDNA
moleculeistwistedintheoppositedirectionasthe
righthandedhelixofB-formDNAaboutitsaxis.

46. Activities of enzymes topoisomerase type I / II and supercoils of DNA
Positive supercoils of DNA Negative supercoils of DNA(
are formed when the DNA molecule is
twisted in the same direction as the right
handed helix of B-form DNA about its axis.
are formed when the DNA molecule is
twisted in the opposite direction as the right
handed helix of B-form DNA about its axis.
Introduced by topoisomerase I and relaxed by
topoisomerase II.
Introduced by topoisomerase II and relaxed
by topoisomerase I.
The amount /activities of enzymes topoisomerase type I and II are regulated to maintain
appropriate degree of negative supercoiling.

47. Supercoils and DNA Topoisomerase
• Super coils are formed as double helix separates from one side & replication
proceed at the other side (twisted ropes pooled apart)
• DNA Topoisomerase Type I –nuclease activity –cuts single strand (to
overcome problem of supercoiling) & reseal the strand by ligase activity.
• DNA Topoisomerase Type II(called DNA Gyrase in prokaryotes): cuts both
strands (to overcome problem of supercoiling) & reseal the strands by Ligase
activity. It introduces negative supercoils to DNA using free energy from ATP
hydrolysis.
❑Cancer treatment
❖Camphotherin –an inhibitor of DNA Topoisomerase Type I
❖Amasacrime & Etoposide- inhibitors of DNA Topoisomerase TYPE II

48. Functions and Mechanism of action of Topoisomerase in DNA Replication:1
Topoisomerase
• Relax torsional strain generated during DNA unwinding by helicases by
causing a transient break in DNA followed by resealing . Topoisomerases bind
covalently to DNA and cleave a phosphodiester bond that is reformed after
the enzyme dissociates from the DNA.
• Removes knots and catalyze catenation(linking together of double stranded,
circular DNA) and decatenation.
• Type I Topoisomerase cleave only one strand and catenate/decatenate
substrate containing a nick ,whereas Type II Topoisomerase enzyme cleaves
both strands of DNA and catenate/decatenate covalently closed cycle.

49. Functions and Mechanism of action of Topoisomerase in DNA Replication:2

50. Functions of Helicases and Topoisomerase in DNA Replication in Prokaryotes
Helicases in conjunction with topoisomerase relieve torsional stain .

51. Able to
covalently link
together
Unable to
covalently link
the 2 individual
nucleotides together
5’
5’
5’
5’
5’
5’
3’
3’
3’
3’
3’
DNA Polymerase Cannot Initiate new StrandsRole of RNA
Primer of DNA
Replication In
Prokaryotes:1
❖ Theprimer:
▪ isrequiredforDNA
synthesis
▪ ShortpieceofRNA(10
nucleotides)
▪ synthesizedbyDNA
dependentRNA
Polymerase (alsocalled
primase)in5’to3’
direction)usingDNAas
template
▪ DNAPolymeraseinitially
addsadeoxynucleotide
to3’OHgroupofthe
primerandthen
continuestoadd
deoxynucleotidesto3’
endofthegrowing
strand.

52. RNA primer is synthesized by
RNA polymerase/primase along with single
stranded binding proteins and forms
primosomes .
Function of RNA Primase of DNA Replication In Prokaryotes

53. DNA primase synthesizes an RNA primer to initiate DNA synthesis on the lagging strand
Synthesis of RNA Primer by DNA Primase
PrimerissynthesizedbyDNA
dependentRNAPolymerase (also
calledprimase)in5’to3’direction)
usingDNAastemplate
DNAPolymeraseinitiallyadds
adeoxynucleotideto3’OHgroup
of theprimerandthencontinues
toadddeoxynucleotidesto3’end
ofthegrowingstrand.

54. Role of RNA Primer of DNA Replication In Prokaryotes:2
❖RNA Primer of DNA Replication In Prokaryotes:
• RNA Primer or initiator RNA ( iRNA ) –A short fragment of RNA 4-12
nucleotides in length base paired to the template DNA and provides free
3’OH group for initiating DNA synthesis by DNA polymerase .
• RNA primer is synthesized by RNA polymerase along with single stranded
binding proteins and forms primosome .
• Leading stand needs a single primer.
• Lagging stand need constant synthesis & supply of RNA primers .
• Extension of RNA Primer is carried out by DNA polymerase III which
functions as a dimer with one core complex of holoenzyme moving
continuously along the Leading stand template while other cycles from one
Okazaki fragment to the other on the Lagging stand .
• RNA Primer is removed by DNA pol I and subsequent gap filling is done by
the same enzyme.

55. Synthesis and replacement of RNA primers during DNA replication

56. DNA polymerases
❖DNA –dependent DNA polymerase catalyze DNA synthesis on DNA
template during replication, following DNA damage ,recombination,
after removal of the primer from the lagging strand.

57. DNA polymerases in E. coli

58. Types of E. coli DNA polymerases
DNA polymerase I
It is also known as
Kornberg enzyme.
It contains 1 or 2
atoms of Zn++ at
active site.
DNA polymerase II
It is minor
component during
growth.
It is induced by SOS
response.
DNA polymerase III
It is the most
active DNA
polymerase.
It has the highest rate
of chain elongation
and Processivity
among E. coli DNA
polymerases.
There are five Types of E. coli DNA polymerases of which polymerase I,II and III studied extensively .

59. Genes coding E coli DNA polymerases
DNA polymerase I
A single
polypeptide
encoded by the
pol A gene
DNA polymerase II
encoded by the
pol B gene
DNA polymerase III
encoded by
pol C, dna E,
dna N, dna q
genes

60. Enzyme activities of E coli DNA polymerases
DNA polymerase I
5’ →3’ polymerase
has both 5’ →3’ and
3’ → 5’exonuclease activities
proteolytic cleavage yields
a large C terminal Kienow
fragment with polymerase
and 3’ → 5’exonuclease
exonuclease activity and a
small fragment with 5’ →3’
exonuclease activities
DNA polymerase II
5’ →3’ polymerase
has 5’ →3’ ’exonuclease
activity and lacks
3’ → 5’exonuclease
activity
DNA polymerase III
5’ →3’ polymerase
has
3’→5’exonuclease
activity

61. Functions of E coli DNA polymerases
DNA polymerase I
Gap filling and
exonuclease
activity ding
replication , repair
and recombination
Nick translation
DNA polymerase II
proofreading
DNA repair
DNA polymerase III
Enzyme functions in
form of a large complex
called DNA polymerase
III with 13 subunits. 
four subunits that
function as a sliding
clamp (increase
Processivity) . The 
complex functions as a
clamp loader.

62. Comparison of E coli DNA polymerases:1

63. Comparison of E coli DNA polymerases:2

64. Comparison of Eukaryotic DNA polymerases

65. DNA polymerases III Of E.coli

66. 5’ →3’ polymerization activity of DNA polymerase
• DNA –dependent DNA polymerase catalyze DNA synthesis on DNA
template by successive addition of dNTPs to the free 3’ OH of growing DNA
chain by following reactions :
• (dNMP)n + dNTP→ (dNMP)n+1 +PPi
• The mechanism involves nucleophilic attack of free 3’ OH group on the
phosphorous atom of incoming dNTP with release of PPi and formation
of diester bonds. PPi is hydrolyzed by pyrophosphatase pulling the
reaction to completion in vivo.
❖The enzyme requires :
1. A primer containing a free 3’ OH group base paired to the template
2. All four dNTPs
3. Mg2+ ions
➢The enzyme has low processivity and cannot synthesis long DNA.

67. Hydrolysis of DNA by nucleases
1. Those enzymes which hydrolyze only from the end of DNA molecule
are called exonucleases.
2. Those enzymes which hydrolyze the internal phosphodiester bonds
of DNA molecule are called endonucleases.
3. certain endonucleases cut at a specific sequence of DNA ; these
molecular scissors are called restriction endonucleases (RE).

68. 5’→3’ exonuclease activity of DNA polymerase
❖This essential for
1. Removal of primer
2. Repair of damaged DNA
3. Excision of nonpaired stench of DNA o a segment containing
chemically modified or mutated nucleotides

69. 3’→5’exonuclease activity of DNA polymerase
❖It serves proofreading function and ensures high fidelity of DNA replication
( only 1 error for ever 10 9 nucleotides or 1 wrong nucleotide /10000 cells)
❖proofreading function removes :
1. A primer terminus not base paired with the template
2. Incorrect nucleotides added to the growing chain
3. Frayed end of a primer cased by partial melting

70. Processivity of DNA polymerase
• Processivity refers to the number of nucleotides added to the nascent
chain before the polymerase dissociate from the template.
• Processivity of DNA polymerase can be increased by an accessory
protein functioning as a sliding clamp that holds the polymerase
firmly while it moves on the DNA and release the enzyme at a region
on the double stranded DNA . Assembly of the clamp requires a
clamp loader.

71. Proof Reading Function of DNA Polymerase III
• Fidelity is maintained by proof reading function of DNA Polymerase III.
• Checks the incoming nucleotide & allows only complementary base
(matched base ) to be added to growing strand.
• Edits its mistakes –if any and removes wrongly added /placed nucleotide.

72. Schematic representation of DNA Polymerase III
Structure resembles a human right hand
Template DNA thread through the palm;
Thumb and fingers wrapped around the DNA

73. Stages of DNA replication
❖The process of replication is divided into 3 stages :
1. Initiation
2. Elongation
3. Termination

74. Initiation of DNA Replication in Prokaryotes

75. Initiation of DNA Replication in Prokaryotes
• Prokaryotic cells lack nuclei and have single circular double stranded
chromosomal DNA . Circular DNA duplex unwinds in such a way as to form an
eye or bubble (replication bubble).
• Initiation of DNA Replication involves unwinding (separation) of two
complementary strands and formation of replication fork.
• Unwinding occurs at a single site with specific DNA sequence on a circular DNA
. This site is called the origin of replication (ori).
• Replication bubble provides two points at which replication occurs (active
synthesis)to form replication fork.
• Replication of double stranded DNA is bidirectional i.e. replication forks move
in both directions away from the origin.
• One round of replication involves synthesis of over 4 million nucleotides in
each new strand and completed within 40 minutes.
• Replication ends at termination point on the other side of DNA .

76. Importance of Replication Bubbles during DNA Replication In Prokaryotes
Bubble is formed –as two complementary strands of DNA separate .
Multiple replication bubbles are formed –essential to enhance replication process .

77. Initiation site (ori)of DNA Replication in Prokaryotes
❖Initiation of DNA Replication in Prokaryotes:
1. DNA Replication in Prokaryotes is initiated at a oriC. (a single site of origin of
replication)
2. Initiation site is a short DNA sequence of 245 base pairs containing four 9
base pair sequences (nanomers) and three A-T rich 13 base pair sequences
(13 –mers) .
3. Many molecules of DNA A (a specific protein) bind to nanomers of a oriC (an
initiation site ) in a cooperative manner with melting of 13 –mers. This
causes separation of two strands of DNA .
4. In Eukaryotes ,there are multiple sites of origin of replication of DNA. These
multiple sites almost exclusively composed of A-T base pairs along the DNA
helix and referred as a Consensus sequence.

78. Initiation site(ori)of DNA Replication in Prokaryotes
❖Two series of short repeats at initiation site of DNA replication in Prokaryotes:
1. three repeats of a 13 bp sequence and
2. four repeats of a 9 bp sequence

79. DNA sequences at the Bacterial origin of Replication
Three A-T rich 13 base pair sequences (13 –mers) initiation site
of DNA replication

80. Steps involved in initiation of DNA Replication in Prokaryotes
• DNA A protein recognizes and binds to the ori of the DNA and successively
denatures the DNA .
• Loading of DNA B(helicase) by DNA C to unwound region of DNA is activated
by DNA A . Release of DNA C from DNA B-DNA C complex after loading.
• DNA Helicases: binds to both replication fork & moves along DNA helix
separating two strands .It is a Zip opener and dependent on ATP for energy
supply.
• Unwinding of DNA occurs bidirectionally by DNA B creating two replication
forks.(Separation of two strands of parent DNA results in formation of
replication fork-Site of active DNA synthesis).
• The replication fork moves along using the parent DNA as a template and the
daughter DNA molecule synthesized.
• Torsional stress induced by unwinding relieved by DNA gyrase/Topoisomerase
by cutting either one or both DNA strands .
• SSB Stabilizes two separated DNA strands and prevent their reassociation.

81. Role of DNA A proteins in Replication at oriC
• DNA replication is initiated by the binding
of DnaA proteins to the DnaA box
sequences
– causes the region to wrap
around the DnaA proteins
and separates the AT-rich
region
Loading of DNA B(a
helicase) by DNA C to
unwound region of DNA
activated by DNA A .
Release of DNA C from
DNA B-DNA C complex
after loading.
Unwinding of DNA
occurs bidirectionally by
DNA B creating two
replication forks.
Separation of two
strands of parent DNA
results in formation of
replication fork(a site of
active DNA synthesis)

82. Formation and functions of primosome
• OncetheDNAstrandsareseparated,thebindingofprimase(DNAGprotein)takesplace.
• Primaseformsacomplexwithproteinsknownasprimosome.Primosomeisformedat
eachofreplicatingforks.
• Functionofprimosome:primosomerecognizeaspecificsiteonDNAwhereRNAprimer
synthesisoccurs.

83. Topoisomerase
Protein complexes of the replication fork
DNA polymerase
DNA primase
DNA Helicase
ssDNA binding protein
Sliding Clamp
Clamp Loader
DNA Ligase
DNA Topoisomerase

84. “Three Dimensional” view of Replication Fork
Direction of fork movement
Direction of synthesis
Of lagging strand
Direction of synthesis
of leading strand

85. Elongation of DNA Replication in Prokaryotes

86. Elongation of DNA Replication in Prokaryotes
• Once each primer has been laid down ,two DNA polymerase III complexes
assembled( one at each of the prime sites).
• Because of antiparallel nature of the two strands , the synthesis of DNA along
the two strands is different.

87. The polarity of DNA synthesis creates an asymmetry between the leading
strand and the lagging strand at the replication fork.
Differential synthesis of DNA along the two strands

88. Elongation of DNA Replication in Prokaryotes
❖ Elongation of DNA Replication in Prokaryotes:
• Two new strands are synthesized in simultaneously in 5’→3’direction by DNA pol III
and in opposite directions . The DNA synthesis can proceed only in 5’→3’direction.
• One strand which runs in the 3’→ 5’ direction .It is replicated continuously by DNA
pol III in the 5’→3’ direction towards replication fork is the Leading strand. This
strand requires only one primer.
• Other strand which runs in the 5’→ 3’ direction .This strand is replicated
discontinuously by DNA pol III in the 5’→3’ direction(in the direction opposite to
the movement of replication fork) . The short fragments of 1 -2 base pairs are
termed as Okazaki fragments. This new strand is known as the Lagging stand . It
needs numerous RNA primers at specified intervals for synthesis of segments of
DNA.
• Thus, RNA Priming is required for synthesis of both Leading stand and Lagging
stand .

89. Elongation of DNA Replication in Prokaryotes

90. Two dimensional view of a replication fork
Direction of synthesis
on leading strand
3’
5’
3’
5’
3’
5’
Synthesis of two
new strands in
simultaneously in
opposite
direction by DNA
Polymerase III
Leading strand : in a direction 5’→3’
towards replication fork &continuous
Lagging strand: a direction
5’→3’ away from
replication fork
& Discontinuous
Synthesis of Leading strand

91. Synthesis of Okazaki fragments in DNA replication of prokaryotes

92. Replication of the Lagging Strand

93. Synthesis of Okazaki fragments in lagging strand during DNA replication of prokaryotes
In Synthesis of Okazaki fragments in DNA replication of prokaryotes
• Incoming Deoxy ribonucleotides are added one after other and to 3’ end of
growing DNA chain.
• Pyrophosphate (Ppi) is removed by addition of each nucleotide.
• Template strand (parent strand)determines the base sequence of newly
synthesized complementary DNA.

94. A 3’ hydroxyl group is necessary for addition of nucleotides
ElongationofDNAstrandduringreplication:IncomingDeoxyribonucleotidesareaddedoneafterotherand
to3’endofgrowingDNAchain.Pyrophosphate(Ppi)isremovedbyadditionofeachnucleotide.

95. Lagging strand synthesis

96. NeedofOkazakifragmentsofLaggingstrandduringDNAReplicationinProkaryotes
• Need of Okazaki fragments of DNA Replication in Prokaryotes is due to
Polarity problem.
• Leading strand : its 3’ end (3’OH) oriented towards the fork therefore
elongation by sequential addition of new nucleotides.
• Lagging strand : no DNA polymerase to add nucleotides to 5’ end of growing
chain (3’→5’ direction )
• In Lagging strand DNA synthesis occurs as series of small fragments called
Okazaki pieces/fragments in normal 5’→3’ direction& later on Okazaki
fragments ligated to form continuous strand.
• Okazaki fragments ligation is done by DNA ligase & DNA polymerase I.

97. Action of DNA Polymerase I
• Upon the completion of lagging and lagging strand synthesis ,the RNA
primers are removed from fragments by DNA polymerase I .
• This polymerase fills the gaps that are produced by removal of the primer .
• But it cannot join two polynucleotide chains together hence additional
enzyme DNA ligase is needed.

98. Replacement of RNA Primer by DNA Polymerase I
• RNA primer is excised by DNA Polymerase I &takes its position.
• DNA Polymerase I catalyzes DNA synthesis in 5’ →3’ direction replaces RNA
primer
• DNA Ligase –catalyzes formation of phospho-diester linkage between DNA
synthesized by DNA Polymerase III and small fragment of DNA produced by
DNA Polymerase I.
• Sealing –requires energy –(ATP→ AMP +Ppi)
• DNA Polymerase II –DNA repair process

99. Synthesis and replacement of RNA primers during DNA replication

100. Functions/ Action of DNA ligase
❖Function of DNA ligase:
1. Repairs single strand nicks in duplex DNA
2. the joining of ends of okazaki fragments
3. Links the ends of linear double – stranded DNA to form circles.
➢This enzyme catalyzes the formation of phosphodiester bond between the 3’
OH group at the end of one DNA chain and 5’ phosphate group ate end of
other.
➢This energy needed for this process is supplied by NAD+ in bacteria and ATP in
eukaryotes.

101. Mechanism of action of ligase in DNA Replication
DNA ligase catalyzes the formation of a phosphodiester bond between two
DNA fragments.
The cofactor for the enzyme is NAD+ in E.coli and ATP in Eukaryotes.
Reaction mechanism :
The enzyme reacts with NAD+ (ATP) to from a covalent adenylyl enzyme
intermediate (E-AMP) and nicotinamide mononucleotide (NMN) PPi.
E+ NAD+ (ATP)  E –AMP + NMN ( PPi )
The adenylyl group is transferred from E –AMP to 5’phosphate of DNA to form
a pyrophosphate bond .
Nucleophilic attack of 5’phosphate by 3’OH group of the adjacent nucleotide
forms a phosphodiester bond.

102. Function of DNA polymerase I and ligase in sealing of okazaki fragments

103. Nick translation
• Nick translation involves degradation of a DNA (RNA) segment by 5’
→3’ ’exonuclease activity of DNA polymerase I , followed by Gap
filling polymerase activity and nick sealing by ligase .This is useful in
radiolabeling nucleic acid strand.

104. Nicks are single strand breaks in double stranded DNA
DNA ligase seals nicks left by lagging strand replication
Nick translation

105. Proofreading Function of DNA Polymerases
❖Fidelity( accuracy) of DNA replication is maintained by proofreading function of
DNA Polymerase III.
❖DNA Polymerase III:
1. Checks the incoming nucleotide & allows only complementary base (matched
base ) to be added to growing strand.
2. Edits its mistakes –if any and removes wrongly added /placed nucleotide.
➢Pol I and Pol II are known to excise nucleotides before the introduction of the
next nucleotide (Proofreading activity).
➢Incorrect nucleotide are incorporated with a frequency 1 in 108 -1012 bases, which
could lead to mutation .The error ratio during replication is kept at a very low level.
➢Mismatches (the incorrect interactions) occur more frequently but do not lead to
stable incorporations because of all three DNA polymerase have 3’ to 5’
exonuclease activity (Proofreading activity) .

106. DNA Polymerase III and DNA Replication of Prokaryotes
❖DNA Polymerase III
• DNA Polymerase III catalyzes DNA synthesis
• Substrate for DNA Polymerase III (Prerequisite for replication) –is four types
of Deoxyribonucleotide triphosphate (dATP ,dGTP, dCTP, dTTP)
• DNA synthesis occurs in 5’ → 3’ Direction ( Anti-parallel to the parent
template strand )
❑Synthesis of two new strands in simultaneously in opposite direction by
DNA Polymerase III
a) One in a direction 5’→3’ towards replication fork &continuous
b) Other in a direction 5’→3’ away from replication fork & Discontinuous

107. Template strand (parent strand)determines the base sequence of newly synthesized
complementary DNA.

108. Termination of DNA Replication in Prokaryotes

109. Termination of DNA Replication in Prokaryotes
❖Termination of DNA Replication in Prokaryotes:
• Two replication forks meet at a specific sequence(termination sequence) of 2
base pairs called Terminator region that bind to a protein factor called Tus (for
Terminus Utilization Substance).
• A specific protein ,Ter binding protein binds to these sequences (Terminator
region) and prevent helicase (DNA B protein) from further unwinding of DNA .
This facilitates the termination of replication.
• Tus- Ter complex arrest replication fork in one direction and other fork halts
when it encounters the arrested replication fork.
• Two topologically interlinked circular chromosomes called catenanes
generated by replication are separated by a type II topoisomerase and
segregated into two daughter cells at cell division.

110. Role of Tus-Ter in Termination of DNA Replication in Prokaryotes
Tus-Tercomplexarrestreplicationforkinonedirection
andotherforkhaltswhenitencountersthearrested
replicationfork.

111. Tus-Ter complex arrest replication fork in one direction
Two replication forks meet at a specific sequence of 2 base pairs called Terminator region
(Ter) that bind to a protein factor called Tus (for Terminus Utilization Substance).

112. Overview
of
termination
of
bacterial
DNA
replication
Twotopologicallyinterlinkedcircular
chromosomescalledcatenanes
generatedbyreplicationare
separatedbyatypeII
topoisomeraseandsegregatedinto
twodaughtercellsatcelldivision.

113. Summary of Functions of enzymes /proteins of DNA synthesis in prokaryotes:1

114. SummaryofFunctionsofenzymes/proteinsofDNAsynthesisinprokaryotes:2

116. Types and functions of Eukaryotic DNA polymerases
Five types of DNA polymerases in Eukaryotes : Pol , Pol , Pol , Pol  and Pol 
❖Pol  :
1. present in the nucleus.
2. has polymerase activity ,lack proofreading function.
3. is involved in the synthesis of short primers that are extended by Pol  .
4. provides the template for replication factor C (RFC) ,an accessory factor that loads DNA
polymerases (similar to E.coli Pol  complex) .Pol  loaded by RFC binds to the initiation
complex at the origin and synthesizes an RNA primer of 1 base pairs followed by 2 -3
base pairs of DNA called initiator DNA (iDNA).The iDNA is then attached by polymerase /.
This is known as pol switch.
❖Pol  :
1. present in the nucleus as a dimer.
2. has low lowest fidelity and processivity.
3. functions in DNA repair.
❖Pol  :is involved in mitochondrial replication .
❖Pol  : is involved in leading strand synthesis and functions in DNA repair.
❖Pol  : is the main enzyme in DNA replication .

117. Functions of Eukaryotic DNA polymerases Pol 
❖Pol  :
1. is the main enzyme in DNA replication .
2. synthesizes both leading and lagging strands .
3. has proofreading function.
4. binds proliferating cell nuclear antigen (PCNA) , an accessory factor
that functions as a sliding clamp and increases processivity.(similar
to  subunit of E.coli Pol III)

118. Comparison of Prokaryotic and Eukaryotic DNA polymerases
Prokaryotic DNA
polymerases
Eukaryotic DNA
polymerases
Functions
I (Pol l)  Gap filling and synthesis between Okazaki fragments of
lagging strand(5’ →3’ polymerization activity)
II(Pol ll)  DNA proof reading and DNA repair(3’→5’exonuclease
activity)
 DNA repair (5’→3’ exonuclease activity)
 Mitochondrial DNA synthesis
III(Pol III)  Functions at replication fork, catalyzing Leading and
lagging strand synthesis

119. Eukaryotic DNA polymerases : Pol ,Poland Pol 
Polbindsproliferatingcellnuclearantigen
(PCNA),anaccessoryfactorthatfunctionsas
aslidingclampandincreasesprocessivity
Polisinvolvedinleadingstrand
synthesisandfunctionsinDNArepair.
Pol  is involved in the synthesis of short
primers that are extended by Pol .

120. Eukaryotic DNA Replication
• In Eukaryotic DNA Replication occurs in the S phase of the cell cycle.
• Eukaryotic DNA Replication is bidirectional occurring at the multiple sites
simultaneously .
• The Replication origins are present in clusters called Replication units. In
human ,there are about 1 ori of replication consisting of 1 base pairs
each.
• Each replicon consist of replication bubbles with two replication forks moving
in opposite directions. Replication continues until the replication bubbles
merge together.
• The mechanism is similar to that seen in prokaryotes.
• There are 5 different types of DNA polymerases which catalyze replication
and repair . (Pol , Pol , Pol , Pol , Pol  )

121. S phase of the cell cycle

122. Prokaryotic and Eukaryotic DNA Replication
In Eukaryotic DNA Replication occurs in the S phase of the cell cycle.
Eukaryotic DNA Replication is bidirectional occurring at the multiple sites simultaneously .

123. Origins in Eukaryotic DNA Replication
The Replication origins of Eukaryotic DNA Replication are present in clusters called
Replication units. In human ,there are about 1
ORI of replication consisting of 1 base pairs each. Each replicon consist of replication
bubbles with two replication forks moving in opposite directions. Replication continues until

124. origins in Eukaryotic DNA Replication
Inyeastcells,theDNAsequenceknownasanautonomouslyreplicatingsequence(ARS)composedalmost
exclusivelyofA-Tbasepairs.ARSisthesitefortheoriginofreplicationcomplex(ORC).

125. Originofreplicationcomplex(ORC)ineukaryoticreplication
❖Originofreplicationcomplex(ORC):madeofsixproteins.
▪ Replicationcomplex:(ORC)sixproteins+licensingfactors
▪ Functionoflicensingfactors:permitstheformationoftheinitiatingcomplex.These
proteinsservetoensurethateachrepliconisreplicatedonceandonlyonceinacellcycle.
▪ Destructionoflicensingfactors:aftertheformationofinitiationcomplex
▪ ProcessofDestructionoflicensingfactors:Destruction ismarkedwhenitistaggedby
Ubiquitinpresentinacell(presentinalleukaryoticcells).Thisisfollowedby
proteasomaldigestionoftheubiquitintaggedproteins.
▪ DNAhelicase: separatestheparentDNAstrandsandarestabilizedbybindingof
replicationproteinA(singlestrandedDNAbindingprotein).
▪ Polymerase : the initiator DNA polymerization(DNA polymerase activity) and
synthesizes RNA primers(primase activity). It lacks exonuclease activity.
▪ Function of protein replication factor C(RFC):displaces of Polymerase  and
attracts proliferating cell nuclear antigen (PCNA).

126. Polymerase switching
• Functions of proliferating cell nuclear antigen (PCNA): PCNA binds to DNA
polymerase  ( function similar to polymerase III of E.Coli). The binding of PCNA
to polymerase  , increases enzyme processivity and starts replicating long
stretches of deoxyribonucleotides .
• This process is called polymerase switching because polymerase  replaces
polymerase .

127. Functions of Polymerase 
❖Functions of Polymerase :
1. DNA polymerization(replicating long stretches of deoxyribonucleotides) .
2. 3’→ 5’ exonuclease activity : edits/repairs the replicated DNA
3. Replication by Polymerase  continues in both directions from the origin of
replication until adjacent replicons meet and fuse .
➢RNA primers are removed by RNase H and the DNA fragments are ligated by
DNA ligase .

128. Functions of RNase H1 and FEN1 during Eukaryotic DNA Replication
• The okazaki fragments in mammals are removed by RNase H1 ,which makes an
endonucleolytic cut , and FEN1 that cleaves primer .
• The newly synthesized DNA is packaged into nucleosomes by proteins termed
as chromatin assembly factors .

129. Replication forks in Eukaryotic DNA Replication
The mechanism of Eukaryotic DNA replication is similar to that seen in prokaryotes.

130. Removal of the okazaki fragments in mammals by RNase H1 (an endonucleolytic cut) and
cleavage of RNA primer by FEN1

131. Sliding clamp of the beta subunit
Pol  which synthesizes both leading and lagging strand binds PCNA ,a cyclin that functions
as a sliding clamp.

132. Chromosomal DNA is packaged and organized at
several levels.
Each phase of condensation
or compaction and organization decreases overall
DNA accessibility to an extent that the DNA sequences
in metaphase chromosomes are almost totally
transcriptionally inert.
In toto, these five levels of DNA compaction result in
nearly a 104-fold linear decrease in end-to-end DNA
length.
Complete condensation and decondensation of the
linear DNA in chromosomes occur in the space of
hours during the normal replicative cell cycle
The newly synthesized DNA is packaged
into nucleosomes by proteins termed as
chromatin assembly factors .

133. End replication problem in Eukaryotic DNA Replication

134. End replication problem in Eukaryotic DNA Replication:1
• In Eukaryotes replicating the ends of the lagging strand becomes a
problem because there is no template available for the RNA primer of
the last Okazaki fragments to bind.
• As a result ,there is an overhang the telomeres with gradual
shortening of the chromosomes after each round of replication.
• Telomeres shortening is considering to be responsible for aging.
• Telomerase, an RNA- protein enzyme complex that carries an RNA
template ,recognizes the G – rich tip of a telomere sequence and
extends it in 5’→3’ direction before replication.
• Consequently ,the 3’ end of the telomere is longer than the 5’ end
and forms a T loop which protects against nuclease attack.

135. End replication problem in Eukaryotic DNA Replication:2
❖Extension of the parental strands by telomerase is followed by:
1. Strand separation
2. Synthesis of new strands
3. Removal of primers
4. Gap filling
The newly synthesized strands that are not shorter than the parental
strands.

136. End replication problem in Eukaryotic DNA Replication:3
Extensionoftheparentalstrandsbytelomeraseisfollowedbystrandseparation,synthesisofnewstrands,
removalofprimersandGapfilling Thenewlysynthesizedstrandsthatarenotshorterthantheparentalstrands.

137. End replication problem in Eukaryotic DNA Replication:4
Afterthenormalreplication,thereisasinglestrandinthisregion,sotheportionisdegradedbyexonucleases.
Thisbrokenendleadstoaberrantrecombinationorendtoendfusions.Unlessthereissomemechanismto
replicatetelomeres,thelengthofthechromosomewillgoonreducingateachcelldivision.Thestabilityofthe
chromosomesisthuslost.Manygenesmightbelostintheprocess.

138. Telomeres

139. Telomeres
Replication always takes place from 5’to 3’ direction in the new strand. The DNA polymerase
enzyme is not able to synthesize the new strand at the end of 5’ end of the new strand . In
other words ,a small portion of ( about 300 nucleotides couldn't be replicated).

140. Importance of Telomeres
• Replication always takes place from 5’to 3’ direction in the new strand. The DNA
polymerase enzyme is not able to synthesize the new strand at the end of 5’ end of the
new strand . In other words ,a small portion of ( about 300 nucleotides couldn't be
replicated).
• This end piece of chromosome is called as Telomeres. Therefore enzyme Telomerase or
Telomere Terminal transferase takes up the job of replication of the end piece of
chromosomes . The Telomeres are noncoding repetitive sequences .
• After the normal replication ,there is a single strand in this region, so the portion is
degraded by exonucleases. This broken end leads to aberrant recombination or end to end
fusions .
• Unless there is some mechanism to replicate telomeres ,the length of the chromosome will
go on reducing at each cell division .The stability of the chromosomes is thus lost. Many
genes might be lost in the process.
• The shortening of Telomeres end is prevented by an enzyme Telomerase. It contains an
RNA component ,which provides the template for telomeric repeat synthesis.

141. Telomeres and aging
Terminalrestrictionfragmentsfrom70yearsoldindividualsareshorterthanthosefrom20yearsold
individuals.Thusinoldage,theTelomeraseactivityislost,leadingtochromosomeinstabilityandcelldeath.
Telomerasemayberesponsiblefortheimmortalizationofcancercells.

142. Characteristics of Telomerase
Telomerase :
▪ is present in microorganisms, plants , animals and germ –line cells of
human .
▪ It acts as a reverse transcriptase .The Telomerase recognizes 3’ end of
telomeres and then a small DNA strand is synthesized.
▪ Terminal restriction fragments from 70 years old individuals are shorter
than those from 20 years old individuals .Thus in old age ,the Telomerase
activity is lost, leading to chromosome instability and cell death.
▪ is absent in normal human somatic cells but present in the cancer cells.
▪ may be responsible for the immortalization of cancer cells.
▪ is a potential target for anticancer agents .

143. Role of Telomerase
The end piece of chromosome is called as Telomeres. Therefore enzyme Telomerase or
Telomere Terminal transferase takes up the job of replication of the end piece of
chromosomes . The Telomeres are noncoding repetitive sequences .

144. Telomerase acts as a reverse transcriptase
Telomerase acts as a reverse transcriptase .The Telomerase recognizes 3’ end of
telomeres and then a small DNA strand is synthesized.

145. Telomerase and human diseases
Telomerase is also implicated in other human diseases which include :
1. Cancer
2. Diabetes mellitus
3. Aplastic anemia
4. Fanconi’s anemia
5. Bloom syndrome
6. Ataxia telangiectasia

146. Telomerase and cancer cells
As a general rule, cancer cells have continued presence of Telomerase and the chromosome
length is maintained ,leading to continued cell division .As the cancer cells have increased and
persistent activity of Telomerase , the cancer cells become immortal .

147. Telomerase and cancer cells
The shortening of Telomeres end is prevented by an enzyme Telomerase. It contains an RNA
component ,which provides the template for telomeric repeat synthesis.

148. The use of antisense oligonucleotides against RNA component of the Telomerase arrests the
uncontrolled cell proliferation with the minimum side effects.
Mechanismofantisense
oligonucleotideagainst
RNA componentto
preventCancercell
proliferation

149. Telomerase and cancer cells
Telomeraseisabsentinnormalhumansomaticcellsbutpresentinthecancercells.Itmayberesponsiblefor
theimmortalizationofcancercells.Itisapotentialtargetforanticanceragents.

150. Telomerase and chemotherapy
➢Telomerase is a therapeutic target for cancer cell chemotherapy .
➢ Inhibition of Telomerase can effectively control the multiplication of
malignant cells .
➢The use of antisense oligonucleotides against RNA component of the
Telomerase arrests the uncontrolled cell proliferation with the minimum side
effects.

151. Telomerase and chemotherapy

152. Telomerase is a target for cancer therapy

153. Comparison of Prokaryotic and Eukaryotic DNA Replication
Features Prokaryotic DNA Replication Eukaryotic DNA Replication
Numberofreplicationorigins Single Multiple
Rate of replication 500 to 1000 nucleotides/sec (10 times
faster than in eukaryotes)
50-100 nucleotides/sec
Types of DNA
polymerases
5(Pol designated by Roman numerals
I,II,III,IV,V)
14(Pol designated by Greek
numerals ,,,, etc.)
RNA primer length 50 nucleotides 9 nucleotides
RNA primer removal DNA pol I RNase H
Nucleotide length
Okazaki fragments of
lagging strand
1000 – 2000 nucleotides 200 nucleotides
Strand elongation DNA pol III DNA pol ,
Telomerase absent Present
Sliding clamp Sliding clamp PCNA

155. Types of DNA damage
❖Types of DNA damage include :
➢Single and double stranded beaks
➢Cross linking between
• Bases in the same strand
• Bases in the opposite strands
• DNA and protein
➢Bases changes such as :
• Insertions
• Deletions
• Covalent modification
➢ substitutions which may be:
• Transitions (replacement of one purine/pyrimidine by another purine/pyrimidine)
• Transversions (replacement of purine by another a pyrimidine or vice versa)
➢Oxidative damage by reactive oxygen species.

156. Causes of DNA damage
❖Causes of DNA damage may be
1. Spontaneous
2. Induced

157. Spontaneous DNA damage
❖Spontaneous DNA damage include :
1. Deamination of cytosine to uracil that base pairs with A instead of G
2. Depurination (apurination)
3. Depyrimidination

158. Spontaneous DNA damage
Deamination of cytosine to uracil that base
pairs with A instead of G

159. Induced DNA damage
❖DNA damage induced by :
1. Radiation
2. Chemicals
➢DNA damage induced by Radiation:
1. Ionizing radiation (X -rays and  -rays) which causes depurination
( apurination) and hydrolysis of phosphodiester bonds.
2. UV radiation from sunlight forms thymine- thymine dimer between
successive T residues, leading to replication errors .

160. Induced DNA damage by radiations
UVradiationfromsunlightformsthymine-thymine
dimerbetweensuccessiveTresidues,leadingto
replicationerrors.
Induced DNA damage by Ionizing radiation
(X -rays and  -rays) on Guanine

161. Induced DNA damage

162. DNA damage induced by Chemicals
❑DNA damage induced by Chemicals
1. Alkylating agents that are strong electrophiles that bind to DNA. Examples
include methylmethane sulphonate (MMS) and ethyl nitrosourea.
2. Non-Alkylating agents such as nitrous acids ,which causes deamination of
cytosine ,Adenine and guanine to uracil, hypoxanthine and xanthine
respectively.
3. Intercalators containing aromatic rings that insert between adjacent base
pairs in DNA e.g. Daunorubicin produced by strains of
Streptomyces(anticancer agent).
4. Base analogs such as bromodeoxyuridine (BroU) which is analog of T
which is incorporated into DNA instead of T. It mispairs with G instead of A,
resulting in AT to GC transition.

163. DNA damage induced by Non-Alkylating agents
Non-Alkylating agents such as nitrous acids ,which causes deamination of cytosine ,Adenine
and guanine to uracil, hypoxanthine and xanthine respectively .

164. Intercalators insert between adjacent base pairs in DNA
Ethidium bromide being fluorescent compound is used for visualization of DNA after its
electrophoretic separation. Daunorubicin(Intercalator) is used as anticancer agent.

165. DNA damage induced by base analogs (Bromodeoxyuridine BroU)
Base analogs such as Bromodeoxyuridine (BroU) which is analog of T which is incorporated
into DNA instead of T. It mispairs with G instead of A, resulting in AT to GC transition.

166. DNA Repair mechanisms

167. Repair of thymine- thymine dimer by photo reactivation
❖Photo reactivation repairs UV induced cyclobutane thymine-
thymine dimers. This is accomplished by the enzyme DNA photolyase,
which contains two chromophores capable of absorbing light.
1. N5N 1 methenyltetrahydrofolyl polyglutamate( MTHF poly Glu)
2. FADH
➢The enzyme DNA photolyase uses energy derived from absorbed
light to reverse damage.

168. DNA Repair by enzyme DNA photolyase using photo reactivation
Two chromophores of DNA photolyase viz. N5N 1 methenyltetrahydrofolyl
polyglutamate(MTHF poly Glu) and FADH are capable of absorbing light. The enzyme DNA
photolyase uses energy derived from absorbed light to reverse damage.

169. Types of Excision Repair for DNA
❖Types of Excision Repair for DNA are :
1. Base Excision Repair for DNA
2. Nucleotide Excision Repair for DNA

170. Base Excision Repair of DNA
❖Base Excision Repair of DNA occurs as follows :
1. Deamination of cytosine ,Adenine and guanine to uracil, hypoxanthine and
xanthine respectively.
2. The bases are removed by DNA Glycosylases to generate apurinic and
apyrimidinic sites.
3. An endonuclease cleaves the a basic sugar and phosphate.
4. The gap is repaired by DNA pol I and sealed by ligase.

171. Base Excision Repair of DNA
ThebasesareremovedbyDNAGlycosylasestogenerateapurinicandapyrimidinicsites.Anendonuclease
cleavestheabasicsugarandphosphate.ThegapisrepairedbyDNApolIandsealedbyligase.

172. Nucleotide Excision Repair for DNA
❖Nucleotide Excision Repair (NER) of DNA : Lesions such as thymine-thymine
dimers , base adducts and 6,4 photoproducts are repaired by Nucleotide
Excision Repair.
• Upto 3 bp of damaged DNA is replaced by Excision of a short stretch of
Nucleotides on either site of the lesions.
• Two phosphodiester bonds on the strand containing defect are hydrolyzed by
an Excision nuclease termed as Excinuclease present in both E.coli and
human . The gap is repaired by DNA pol I in E.coli and pol / in human.
• The Nick is sealed by DNA ligase.

173. Nucleotide Excision Repair for DNA
NucleotideExcisionRepair(NER) ofDNA:Lesionssuchasthymine-thyminedimers,baseadductsand6,4
photoproductsarerepairedbyNucleotideExcisionRepair.

174. Nucleotide Excision Repair for DNA in E.Coli and Human
Upto3bpofdamagedDNAisreplaced byExcisionofashortstretchof Nucleotidesoneithersiteofthe
lesions.TwophosphodiesterbondsonthestrandcontainingdefectarehydrolyzedbyanExcisionnuclease
termedasExcinucleasepresentinbothE.coliandhuman.ThegapisrepairedbyDNApolIinE.coliandpol/
inhuman. TheNickissealedbyDNAligase.

175. Repair mechanism for DNA mismatches :1
❑The DNA mismatch repair (MMR) system corrects errors made during DNA
replication to ensure high degree of fidelity of replication .
❑ DNA mismatches arise due to
1. Mispairing
2. Insertion of extra unpaired bases
3. Deletion as a result of polymerase slippage
Before DNA mismatches are repaired, the parental (template ) strand is
methylated on the N5 position of all adenine residues within the 5‘GATC3’
sequences by Dam methylase in order to differentiate from newly synthesized
strand .
The DNA mismatch repair is targeted to the transiently unmethylated newly
synthesized strand.
This system repairs up to 1 bp from hemi methylated GATC sequence.

176. Strand directed mismatch repair for DNA in E.Coli
• Specific proteins scan the newly synthesized DNA Strand (new DNA
Strand is identified by not being methylated).
• The mismatch area is identified ,a loop is made.
• In E.coli ,this recognition and looping is made by three
proteins viz MutS, MutH , MutL .Then that segment are removed.
Finally the correct segments are synthesized by DNA polymerase,
SSBs and ligase .

177. Repair mechanism for DNA mismatches in E.Coli
In E.coli ,the proteins required to recognize the mutation and catalyze nicking
include MutS, MutH , MutL .
MutS recognizes mismatch and repairs it.
MutH attaches to MutS by MutL and cleaves unmethylated newly synthesized
strand by site specific endonuclease activity marking strand for repair .
An endonuclease degrades the unmethylated strand containing mutation from
the GATC through the mutation to remove damaged DNA.
If mutation is present between two GATC sites ,the DNA between two sites
removed, the gap is filled by polymerase and nick is sealed by ligase .
Several thousands of bases may be replaced by in ode to correct one
mismatch.

178. Strand directed mismatch repair of DNA in E.Coli
The mismatch area is identified ,a loop is made. In E.coli ,this recognitionand looping is made by
three proteinsviz MutS,MutH, MutL.Thenthatsegmentareremoved.Finallythecorrectsegmentsare
synthesizedbyDNApolymerase,SSBsandligase.

179. Repair mechanism for DNA mismatches in human
❖Repair mechanism for DNA mismatches in human:
➢hMSH2 and hMLH 1 are human analogs of E.coli MutS, and MutL .
➢ In human defective mismatch repair in Hereditary nonpolyposis
colon cancer (HNPCC) is associated with microsatellite instability .

180. Transcription coupled Repair
❑Transcription coupled Repair :
The template strand of transcribed region responds immediately by stalling of
RNAP at the site of damage .The enzyme backs away. The Repair proteins are
recruited and the defect is repaired by NER.
❖ Cockyane ’s syndrome results from deficiency of proteins that help to
recognize the halted RNAP.

181. Role of RPNA and Repair proteins at the DNA damage site in human
Transcription coupledRepair:ThetemplatestrandoftranscribedregionrespondsimmediatelybystallingofRNAPatthesiteof
damage.Theenzymebacksaway.TheRepairproteinsarerecruitedandthedefectisrepaired byNER.

182. Failure of Transcription coupled Repair in Cockyane’ssyndrome
ThetemplatestrandoftranscribedregionrespondsimmediatelybystallingofRNAPatthesiteofdamage.The
enzymebacksaway.TheRepairproteinsarerecruitedandthedefectisrepaired byNER.Cockyane’s
syndromeresultsfromdeficiencyofproteinsthathelptorecognizethehaltedRNAP.

183. ClinicalmanifestationsofCockyane’ssyndrome

184. Summary of Repair mechanisms for DNA
Mechanisms for DNA Repair Defect Repair
1.Mismatch Repair Copying error 1-5
bases unpaired
strand cutting and
endonuclease digestion
2.Nucleotide Excision Repair for DNA Chemical damage to a
segment
30 bases removed and then
correct bases added
3.Base Excision Repair Chemical damage to
single base
bases removed by N –
glycosylase and then new
bases added
4.Double strand break free Radicals and
radiation
Unwinding , alignment
,ligation

185. Types of DNA repair systems in E.Coli
DNArepairsystemsinE.Coli TypeofDNAdamage EnzymesandproteinsinvolvedinDNA
repair
Mismatchrepair Mismatchescopyingerrors DNAmethylase
MutH,MutL,MutSproteins
DNAhelicaseII
SSB
DNApolymeraseIII
ExonucleaseI
DNAligase
Baseexcisionrepair Spontaneous,chemicalorradiationdamagetoa
singlebase,Pyrimidinedimers,alkylatedbases
DNAglycosylases
APendonucleases
DNApolymerasesI
DNAligase
Nucleotideexcisionrepair DNAlesionsthatcauselargestructuralchanges e.g.
pyrimidine dimersmaybeduetochemical
,radiationorspontaneous.
ABCExcinuclease
DNApolymeraseI
DNAligase
Directrepair Pyrimidinedimers,O6 –methylguanine DNAphotolyases,O6 –methylguanine

186. Summary of DNA damage and repair

187. Consequences of unsuccessful DNA damage repair
1. Cell death due to Mutations→ Apoptosis , Cell necrosis
2. Cell which survives after mutation→ premature differentiation ,malignant transformation, senescence

188. Diseases associated with defective DNA Repair mechanisms
Diseases Defectss
Xerodermapigmentosa(XP) Defective NucleotideExcisionRepair(NER) forDNA,sensitivitytoUVlight,skin
cancer
AtaxiaTelangiectasia DefectiveATMgene,sensitivitytoUVlight,lymphoreticularneoplasm
Fanconi’sanemia Defectivegenesareinchromosome20qand9q.defectinDNAcrosslink
Repair
Bloom’ssyndrome geneareinchromosome15q,defectinDNAligaseorhelicase,
lymphoreticularmalignancies
Cockyane’ssyndrome DefectiveNucleotideExcisionRepair(NER) forDNA,transcriptionfactorIIHis
defective,stuntedgrowth,mentalretardation
Hereditarynonpolyposis
coloncancer(HNPCC) =
Lych’ssyndrome
Defectivegene inchromosome2.DefectinhMSH1and2genes,mismatch
RepairmechanismforDNAisdefective.

189. Xeroderma pigmentosa
• DNA is constantly being subjected to environmental insults that cause the
alteration of nucleotide bases.
• Exposure of cells to UV light can result in covalent joining of adjacent thymine
(thymine-thymine dimer) . Luckily ,cells are remarkable efficient at repairing
the damage done together DNA.
• In a rare genetic disease Xeroderma pigmentosa the thymine dimer formed in
DNA after skin cells are exposed to UV light and cannot be repaired. Patients
suffer from skin cancer .The enzyme required in repair mechanism ,viz UV
specific endonuclease is absent in these individuals .

190. Xeroderma pigmentosa
InararegeneticdiseaseXerodermapigmentosathethyminedimerformedinDNAafterskincellsare
exposedtoUVlightandcannotberepaired.Patientssufferfromskincancer.Theenzymerequiredinrepair
mechanism,vizUVspecificendonucleaseisabsentintheseindividuals.

191. Ataxia Telangiectasia
❖Ataxia Telangiectasia :
1. Common autosomal recessive disease
2. sensitivity to UV light
3. Cerebellar ataxia
4. Telangiectasia in eyes
5. lymphoreticular neoplasm
6. Ataxia Telangiectasia mutated (ATM) gene located at chromosome 11 q
mutated (ATM gene 1% of total population).
7. Disease is manifested in 1:40000 persons.

192. Ataxia Telangiectasia mutated (ATM) gene located at chromosome 11 q

193. Clinical manifestations of Ataxia Telangiectasia
Commonautosomalrecessivedisease,sensitivitytoUVlight,Cerebellarataxia,Telangiectasia(spiderveins)
ineyes,lymphoreticularneoplasm

194. Mutations
• Mutations are some defects remaining in DNA after proofreading in DNA
replication .
• Mutations may result from faulty DNA replication or repair.
• Mutation rate is 6 nucleotide changes /year in the germ cell lines of an
individual .
• Out if every 10 6 cell divisions ,one Somatic Mutation taking place .

195. Mutations
❖Mutation is a change in the nucleotide sequence of a gene ( a portion
DNA ).
❖Mutation will result in substitution of different amino acid at the
corresponding site in the protein molecule.
❖The altered protein may
1. have a function similar to the normal protein.
2. have a partial but abnormal function or may
3. not be capable of functioning at all.
These three possibilities are exemplified by hemoglobinopathies *.
❖Natural Mutations are responsible not only for genetic disorders ,but
also for the evolutions of newer and novel forms of life .This is suggested
to be responsible evolution.

196. Mutations *related hemoglobin synthesis
Functional Status
of hemoglobin
Proteinhemoglobin Aminoacidsubstitution Codons
function not
altered
Hb ,  chain
Hb Hikari
61 Lysine
Asparagine
AAA or AAG
AAU or AAC
function partially
altered
Hb ,  chain
Hb S
6 Glutamic acid
Valine
GAA or GAG
GUA or GUG
Non Functional Hb ,  chain
Hb M ( Boston)
58 Histidine
Tyrosine
CAU or CAC
UAU or UAC

198. Inhibitors of DNA Replication as antibacterial agents
Some drugs inhibit bacterial enzymes and they will arrest new DNA synthesis
and arrest the bacterial cell division. These drugs are useful as antibacterial
agents(will not affect human cells).
Antibacterial agents (Drugs) functioning as the Inhibitors of DNA Replication enzyme -
Bacterial DNA gyrase
Ciprofloxacin
Nalidixic Acid
Novobiocin

199. Ciprofloxacin as Antibacterial agent (Drug) functioning an inhibitor of DNA
Replication enzyme -Bacterial DNA gyrase
CiprofloxacininhibitssbacterialDNA gyrase enzyme.ItwillarrestnewDNAsynthesisandarrestthe
bacterialcelldivision.Ciprofloxacin, Nalidixic Acid and Novobiocin areusefulasantibacterialagents(will
notaffecthumancells).

200. Inhibitors of DNA Replication as anticancer agents
Some drugs inhibit human enzymes and the will arrest new DNA synthesis
and arrest the cell division. These drugs are useful as anticancer agents .
Anticancer agents(Drugs) functioning as Inhibitors of human DNA Replication
enzyme(DNA Topoisomerases)/DNA polymerase/thymidylate synthase
Doxorubin inhibit human DNA Topoisomerases
Andriamycin inhibit human DNA Topoisomerases
Etoposide inhibit human DNA Topoisomerases
5-Fluorouracil inhibit human DNA polymerase
Mercaptopurine inhibit thymidylate synthase

201. Doxorubinfunctioningas InhibitorofhumanDNAReplicationenzyme(DNATopoisomerases)

202. Mechanism of action of 5-Fluorouracil as an Anticancer agent(Drug)
Antimetabolitessuchas5-FluorouracilandmethotrexateinhibittheformationofdNTPs,thesubstrateforreplication.

203. Inhibitors of DNA Replication
❖Certain nucleotide analogues like arabinose cytosine and arabinose
adenine which contain Arabinose ( another pentose) instead of the usual
ribose inhibit synthesis of DNA.
• Arabinose cytosine : used as an anticancer agent
• Arabinose adenine : used as an antiviral agent
❖Antimetabolites such as 5-Fluorouracil and methotrexate inhibit the
formation of dNTPs, the substrate for replication.
❖Substrate analogs are incorporated into DNA polymerization .These include
a) Azothymidine (AZT ,zidovudine) used in treatment of human
immunodeficiency virus (HIV) infection
b) Cytosine Arabinoside (cytarabine),an antileukemia drug.
❖ Direct Inhibitors of DNA Replication include

204. Mechanism of action Cytosine Arabinoside as an antileukemia drug
Cytosine Arabinoside is a substrate analogs .It is incorporated into DNA and inhibits DNA
replication/ polymerization. Thus it arrests cell division(functions an antileukemia drug).

205. Direct Inhibitors of DNA Replication
❖ Direct Inhibitors of DNA Replication include:
• Intercalators containing aromatic rings that insert between adjacent base
pairs in DNA . These include anticancer agents such as daunorubicin,
produced by strains of Streptomyces.
• DNA damaging drugs including
a) Alkylating agents that are strong electrophiles that bind to DNA.
Examples include methylmethane sulphonate (MMS) and ethyl
nitrosourea.
b) Platinum coordination complexes such as cisplatin that interact with the
N7 Guanine in DNA to form crosslinks between adjacent Guanines.
c) Bleomycin produced by Streptomyces verticillus cause DNA damage by
interacting with O2 and Fe 2+ .These are used as cancer chemotherapeutic
drugs .

206. Synthetic drugs as Inhibitors of DNA Replication in Prokaryotes
• Mitomycin C , Novobiocin : Inhibitors of DNA replication enzyme DNA
polymerase and hence cell division→ Inhibit cell division)
• Ciprofloxacin, ,Nalidixic Acid (Mechanism of action : inhibition of DNA
Topoisomerase II(DNA Gyrase )→DNA replication inhibited → bacterial
cells multiplication stops.
• 5 Fluorouracil & Mercaptopurine: nucleotide analogs→ used cancer
treatment
• Doxorubin ,Andriamycin ,Etoposide : inhibit human DNA Topoisomerases
→ used cancer Treatment

207. Inhibitors of DNA Replication enzymes
Inhibitors of DNA Replication enzymes such as nalidixic acid and
fluoroquinolone are used in the treatment of urinary tract infections .
These inhibit bacterial topoisomerase II( DNA gyrase). It prevents from
relieving positive supercoils during DNA Replication.

208. Plasmids
• Plasmids are small circular DNA molecules present in some bacteria
besides their large circular chromosomal DNA.
• chromosomal DNA of bacteria has complete set of genes for the
existence and propagation of the bacteria. 
• Plasmids have sufficient genetic information to replicate in the
bacterial cells and synthesize few proteins .
• Some naturally occurring bacterial plasmids code for enzymes
proteins that can protect the cells from bactericidal effects of
antibiotics. Thus bacteria which have such plasmids have an
advantage over other bacteria to survive even in the presence of
antibiotics .

209. Plasmids

Plasmids are relatively smaller DNA molecules ,they can be easily isolated from bacterial cell debris and chromosomal DNA
with considerable ease .Therefore Plasmids are used as carriers / vectors of DNA segment from an other source to generate
recombinant DNA . Some naturally occurring bacterial plasmids code for enzymes proteins that can protect the cells from
bactericidal effects of antibiotics. Thus bacteria which have such plasmids have an advantage over other bacteria to survive
even in the presence of antibiotics .

210. Use of Plasmids in molecular biology
• Since Plasmids are relatively smaller DNA molecules ,they can be
easily isolated from bacterial cell debris and chromosomal DNA with
considerable ease .Therefore Plasmids are used as carriers / vectors
of DNA segment from an other source to generate recombinant DNA .
• Such recombinant DNA could be replicated /multiplied / generated in
bacterial cells in large quantities for DNA sequencing ,mutagenesis
and other molecular biology experiments .

211. Plasmid Vectors as the tool for genetic engineering
Recombinant DNA could be replicated /multiplied / generated in bacterial cells in large
quantities for DNA sequencing ,mutagenesis and other molecular biology experiments .