DNA Replication

1. Asst. Professor
Dept. Of Biochemistry,
DS Medical College, Perambalur
Ashok Katta
By
Replication

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3. RNA
DNA
Protein
Transcription
Translation
Replication
Reverse
Transcription
The flow of information in the cell starts at DNA, which replicates to form more DNA.
Information is then ‘transcribed” into RNA,
and then it is “translated” into protein.
The proteins do most of the work in the cell.

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Each replicated duplex
daughter DNA molecule
contains one parent strand
and one newly synthesized
strand.
The base pairing rule is
always maintained.

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Replication in prokaryotes is much better
understood than replication in eukaryotes.
The basic requirements and components of
replication are the same for prokaryotes as for
eukaryotes.

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11. The four deoxyribonucleoside triphosphate.
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12. Both the strands of DNA double helix serve as
template for the synthesis of new daughter
DNA strands.
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Helicase
DNA
Polymerase
Primase
Ligase
DNA Replication Key Enzymes

14. DNA B protein (Helicase)
• Unwinds DNA
Primase
• Synthesizes RNA primer
DNA topoisomerase I & II
• Relieves torsional strain by cutting &
joining single strand or both strands. 17

15. DNA polymerase
• DNA chain elongation
DNA ligase
• Joins Okazaki fragments
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16. DNA A protein
• Opens duplex at origin of replication
SSB (Single strand binding protein)
• Binds separated single stranded DNA and
stabilizes it.
Ter binding protein
• Prevents the helicase from further
unwinding and facilitates termination
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17. DNA protein B or Helicase unwind the DNA
double helix.
It acts like zipper to unzip the DNA.
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18. The DNA synthesis is catalyzed by enzyme
called DNA dependent DNA polymerase.
These polymerases required for:
– DNA chain elongation
– DNA repair (5' – 3' exonuclease activity)
– Proofreading (3' – 5' exonuclease activity).
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Prokaryotic
polymerase
Eukaryotic
polymerase
Functions
I 
Gap filling & synthesis between
Okazaki fragments of lagging
strand
II  DNA proofreading & DNA repair
 DNA repair
 Mitochondrial DNA synthesis
III 
Leading and lagging strand
synthesis

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Primer is short piece of RNA (5–50 NTes in
length) require for synthesis of DNA.
Synthesized by DNA dependent RNA polymerase
called Primase (in a 5’ to 3’ direction) using DNA
as a template.
The enzyme primase in association with SSB
proteins forms a complex called primosome, &
produces RNA primers.

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•
Binding of Helicase
•
Origin of Replication
Ori
• Replication bubble/eye
New strandParent strand

26. In eukaryotes replication begins at multiple
sites composed almost exclusively of A-T base
pairs along the DNA helix and is referred to as a
consensus sequence.
One round of synthesis involves over 4 million NT in each strand of
DNA which complete in about 40 min.
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…

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…

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This unwinding leads to the formation of “V” or
“Y” shaped structure called replicating fork.
SSB stabilizes the separated strands & prevent
their re-association.
The stress produced by supercoiling is released
by topoisomerases.
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33. Once the strands are sufficiently separated,
binding of primase takes place.
And forms complex with proteins called
primosome which can recognise specific
site for synthesis of RNA primer.
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36. 1. First DNA A protein recognizes and binds to the
“ori” of the DNA and successively denatures the
DNA.
2. DNA B protein (helicase) then binds to this
region and unwinds the parental DNA, and form
a “V” where active synthesis occurs. This region
is called the replicating fork.
3. The stress produced due to unwinding by
helicase is released by topoisomerases by
cutting either one or both DNA strands.
4. The Single stranded binding (SSB) protein
stabilizes the separated strands and prevents
their reassociation. 39

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Topoisomerase
Helicase (DNA B protein)
SSB
Direction of movement
Of replication fork
Okazaki fragments
Lagging strand
Leading strandRNA primer
RNA primer
3’
5’3’
5’
3’ 5’
5’
5’
5’
3’
3’

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42. DNA polymerase III initiates the synthesis of new
DNA strand by adding deoxyribonucleotide to the
3' end of the RNA primer.
Thus, DNA polymerase III can synthesize a new
chain only in the 5' to 3' direction.
Both the DNA strands are synthesized
simultaneously but in opposite direction one is in
direction towards the replication fork, the other in
a direction away from the replication fork.
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Topoisomerase
Helicase (DNA B protein)
SSB
Direction of movement
Of replication fork
Okazaki fragments
Lagging strand
Leading strandRNA primer
RNA primer
3’
5’3’
5’
3’ 5’
5’
5’
5’
3’
3’

44. The DNA chain which runs in the 3' → 5'
direction is copied by polymerase III as a
continuous strand, requiring one primer.
This new strand is known as the leading
strand.
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45. The DNA chain which runs in the 5’ → 3'
direction is copied by polymerase III as a
discontinuous manner because synthesis can
only proceed in the 5' to 3' direction.
This new strand is known as the lagging strand.
This requires numerous RNA primers.
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47. As the replication fork moves on lagging
strand, RNA primers are synthesized at
specified intervals.
These RNA primers are extended by DNA
polymerase III into short pices of DNA
called Okazaki fragments.
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48. Okazaki fragments are short, newly synthesized
DNA fragments that are formed on the lagging
template strand during DNA replication.
Okazaki fragments are between 1000 and 2000
nucleotides long in Prokaryotes and are
approximately 150 nucleotides long in eukaryotes.
They are separated by ~10-nucleotide RNA
primers.
Two Okazaki fragments are connected into one
continuous newly synthesized complementary
strand by enzyme ligase.

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50. Upon completion of lagging strand synthesis,
the RNA primers are removed from fragments
by DNA polymerase I.
It also fills the gaps that are produced by
removal of the primer leaving only a nick.
DNA Polymerase I cannot join two
polynucleotide chains together

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52. As DNA Polymerase I cannot join two
polynucleotide chains together, an additional
enzyme is requires for an additional enzyme
DNA ligase is required to perform this
function.
DNA ligase join two polynucleotide chains
together by catalysing the formation of a
phosphodiester bond to seal the Okazaki
fragments.
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55. Termination sequences, e.g. “ter”, direct
termination of replication.
A specific protein, ter binding protein, binds
these sequences and prevents the helicase
from further unwinding of DNA and
facilitates the termination of replication.
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57. DNA is copied by DNA polymerase with high fidelity
(accuracy).
Incorrect nucleotides are incorporated with a
frequency of one in 108–1012 bases, which could lead
to mutation.
But the error ratio during replication is kept at a very
low level by specific process called proofreading.
Mismatches, occur more frequently but do not lead to
stable incorporations because the all three DNA
polymerases have 3' to 5' exonuclease activity
(proofreading activity).
DNA polymerase I & II are known to excise mismatched
nucleotides before the introduction of the next
nucleotide.

60. Some antibacterial, antiviral and many
chemotherapeutic drugs inhibit REPLICATION.
Many antibacterial drugs like Novobiocin, Nalidixic
acid & Ciprofloxacin inhibit the prokaryotic
topoisomerase (DNA gyrase).
These topoisomerase inhibitors are widely used for
the treatment of urinary tract and other infections.
Camptothecin, an antitumor drug, inhibits human
topoisomerase.
Certain anticancer & antiviral drugs inhibits elongation
of DNA chain by incorporating certain nucleotide
analogs, e.g. 2, 3 deoxyinosine.
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62. RNA primer length ~50 nucleotides 9 nucleotides
DNA polymerase
Three types
(I, II, III)
Five types
(α, β, γ, δ and ε)
Number of origins Single Multiple
Nucleotide length
of Okazaki
fragments of
lagging strand
1000–2000
nucleotides
~200 nucleotides
Rate of replication ~500 NTs per sec
50 NTs per sec
(10 times slower than
prokaryotes)

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