3. Identical
base sequences
5’
5’
3’
3’ 5’
5’
3’
3’
WatsonandCrickdouble-strandedDNAstructure
Twisted 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. Organization of Eukaryotic DNA
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. 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
Transcription
RNA
Translation
Protein
Reverse Transcription
Replication
The flow of genetic information
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
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
In semi conservative mechanism , each
replicated duplex daughter DNA
molecules contains one parent strand
and one newly synthesized strand.
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.
22. DNA Replication in Prokaryotes: Semiconservative :
half of original DNA is conserved in the daughter DNA .
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.
27. Overview of bacterial DNA replication
Twotopologicallyinterlinkedcircular
chromosomescalledcatenanes
generatedbyreplicationare
separatedbyatypeII
topoisomeraseandsegregatedinto
twodaughtercellsatcelldivision.
28. Directionality of the DNA strands at a replication fork
Anew strand of DNAis always
synthesized in the 5==3 direction,
with the free 3 OH as the point at
which the DNAis elongated
Fork movement
Lagging strand
Leading strand
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 .
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.Bothcorecomplexandclampdissociateaftersynthesis
ofOkazakifragmentsandagainassociatethenextOkazakifragment.
43. Role of clamp loader
A “clamp loader:” complex is required to get the clamp onto the DNA.
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.
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’
3’
5’
5’
3’
5’
5’
5’
3’
3’
3’
DNA Polymerase Cannot Initiate new Strands
Role 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.
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.
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’exonucleas
e 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.
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
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 A
T-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
DNATopoisomerase
84. “Three Dimensional” view of Replication Fork
Direction of fork movement
Direction of synthesis
Of lagging strand
Direction of synthesis
of leading strand
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 .
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
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.
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
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
DNAligase 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.
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.
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 ,Poland Pol
Polbindsproliferatingcellnuclearantigen
(PCNA),anaccessoryfactorthatfunctionsas
aslidingclampandincreasesprocessivity
Polisinvolvedinleadingstrand
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 )
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).
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 DNAis packaged and organized at
several levels.
Each phase of condensation
or compaction and organization decreases overall
DNAaccessibility to an extent that the DNAsequences
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 DNAin 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 .
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.
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.
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.
