Synthesis of DNA, inhibitors of replication and reverse transcription

1. Replication
R. C. Gupta
Professor and Head
Department of Biochemistry
National Institute of Medical Sciences
Jaipur, India

2. Nucleic acids are known as information
molecules
They are of two types − deoxyribonucleic
acid (DNA) and ribonucleic acid (RNA)
DNA stores genetic information

3. The total genetic material present in an
organism is known as its genome
When a cell divides, the genomic infor-
mation is passed on to the daughter cells
The DNA transmitted to daughter cells is
an exact replica of the DNA of parent cell

4. Parent DNA
New DNA New DNA
Identical base sequence

5. Since new DNA is a replica of parent
DNA, DNA synthesis is called replication
For replication, the two strands of DNA
separate
Each strand serves as a template for the
synthesis of a new strand
Replication – An overview

6. The new strand is complementary in base
sequence to its template strand
The template strand and new strand wind
around each other
Thus, the new DNA is made up of one
parent strand and one new strand
Therefore, replication is said to be semi-
conservative

7. Parent
DNA
Strand
separation
New
DNA
New
DNA
Semi-conservative replication of DNA

8. Replication has been studied extensively
in prokaryotes
Much of the information has been
obtained from E. coli
Eukaryotic replication is more complex but
there are many common features
Replication

9. For replication, DNA has to uncoil and the
strands have to separate
Separation of strands is known as melting
or denaturation of DNA
This requires breaking of hydrogen bonds
between base pairs i.e. A & T and G & C
Prokaryotic replication

10. There are two hydrogen bonds between
A & T and three between G & C
Melting of DNA is easier in those regions
where A & T pairs are more in number
A
− S − P − S − P −S − P −
− S − P − S − P −S − P −
A
T
T
G
C

11. Replication of DNA begins at a specific
site, called origin of replication (ori)
Origin of replication is a site rich in A&T
pairs
This makes strand separation easier

12. In E. coli, the origin of replication (oriC) is
a 245-bp sequence
There are three tandem repeats of a 13-
bp sequence
There are four copies of a 9-bp sequence

13. oriC
(245-bp)
Tandem 13-bp sequences
(GATCTNTTNTTTT)
Four 9-bp sequences
(TTATNCANA)
Origin of replication in E.coli

14. In prokaryotes, there is only one origin of
replication
Separation of strands at this site produces
a fork-like structure
This is known as replication fork
Replication fork

15. Replication fork

16. Both the strands of DNA are replicated
simultaneously
The strands are anti-parallel i.e. they run
in opposite directions
However, nucleic acids are synthesized
only in 5’ 3’ direction

17. One DNA strand is known as the leading
strand
The other strand is known as the lagging
strand
Leading strand is replicated continuously
Lagging strand is replicated discontinuously

18. The replicating enzyme first replicates the
leading strand
Then it turns back and replicates the
lagging strand
Thus, the overall direction remains 5’ 3’

19. Movement
of fork
New strand (continuous)
New strand (discontinuous)
Parent strands
5’
5’
5’
5’
3’
3’
3’
3’

20. DNA is a polymer of deoxyribonucleotides
Deoxyribonucleotides are polymerized by
DNA polymerase
Primers and Okazaki fragments
EMB-RCG
However, DNA polymerase has a major
limitation

21. EMB-RCG
DNA polymerase cannot add a nucleotide
to another nucleotide
It can add a nucleotide only to an
oligonucleotide

22. DNA polymerase cannot
add a nucleotide to another
nucleotide
DNA polymerase can add a
nucleotide only to an oligo-
nucleotide
Mononucleotide
Mononucleotide
Mononucleotide
Oligonucleotide

23. Thus, the first requirement for DNA
synthesis is to form an oligonucleotide
DNA polymerase cannot synthesize the
oligonucleotide
To solve this problem, the synthesis of
DNA begins with ribonucleotides

24. RNA polymerase can add a nucleotide to
another nucleotide
Ribonucleotides are polymerized by RNA
polymerase
RNA polymerase differs from DNA poly-
merase in one important respect

25. Hence, some ribonucleotides are poly-
merized first to form an RNA primer
Synthesis of RNA primer is catalysed
by primase
Primase is an RNA polymerase

26. RNA primer is an oligonucleotide
DNA polymerase can add deoxyribonucleo-
tides to this oligonucleotide
Thus, RNA primer is required to initiate
replication

27. The substrates for primer synthesis are
ATP, GTP, CTP and UTP
The a-phosphate of new ribonucleotide
binds to 3’-OH group of the previous one
The b- and g-phosphates are split off in
the form of inorganic pyrophosphate

28. RNAP
OH
H
OH
H
HH
O
CH2
O
P
O
H
OH
H
HH
O
P ~ P
O
P
OH
H
OH
H
HH
OO
P
Basen+1
3’
OH OH
H H
H
O
P ~ P ~ P – O– CH2
Formation of phosphodiester bond between the last
nucleotide and the new nucleotide
abg
CH2
CH2
Basen+1
Basen
Basen
5’
5’
5’
5’
3’
3’
3’
New nucleotide
Last nucleotide
H

29. Selection of nucleotides is governed by
the base sequence of template strand
The nucleotides are selected according
to the base-pairing rule i.e.
Adenine opposite thymine
Uracil opposite adenine
Guanine opposite cytosine
Cytosine opposite guanine

30. After formation of RNA primer, deoxyribo-
nucleotides are added by DNA polymerase
The substrates are dATP, dGTP, dCTP and
dTTP
The reaction is similar to the addition of
ribonucleotides
This process continues until about 100
deoxyribonucleotides have been added

31. The resulting polynucleotide is known as
an Okazaki fragment
It is made up of a short RNA primer and
several deoxyribonucleotides
Okazaki fragment
3’5’
Primer Deoxyribonucleotides

32. Several Okazaki fragments are formed
on the template strand
3’
DNA template
3’
Okazaki fragments
5’
5’
After replication of several leading and
lagging strands, RNA primers are removed

33. Ribonucleotides are removed one by one
from the 5’-end
They are replaced by deoxyribonucleotides
according to the base-pairing rule
The remaining portions of parent DNA are
then replicated
The different DNA fragments are sealed
together

34. Each new strand forms a duplex with
the template strand of the parent DNA
The winding begins even while the
replication is going on

35. Enzymes and proteins involved in
prokaryotic replication
The process of replication requires a
number of enzymes and protein factors
In E. coli, the unwinding of parent DNA is
initiated by dna B protein

36. First, dna A protein binds to the DNA at
the initiation site
Then, it is joined by dna B and dna C
proteins
The dna B protein begins the unwinding

37. As replication proceeds, further unwinding
is catalysed by helicase (rep protein)
ATP is required as a source of energy for
unwinding DNA
The unwound strands are held apart by
single strand binding (SSB) protein

38. Synthesis of RNA primer is catalysed by
primase
Deoxyribonucleotides are added to the
RNA primer by DNA polymerase
There are three DNA polymerases in
E. coli

39. Arthur Kornberg had discovered the first
DNA polymerase (DNA polymerase I)
Arthur Kornberg
DNA polymerase II and DNA polymerase
III were discovered later

40. Deoxyribonucleotides are added to RNA
primer by DNA polymerase III holoenzyme
DNA polymerase III possesses two different
catalytic activities:
Polymerase activity
3’ 5’ Exonuclease activity

41. The polymerase activity adds deoxyribo-
nucleotides
The 3’ 5’ exonuclease activity is meant
for proof-reading and error-correction

42. The enzyme moves along the template
strand
The polymerase activity add a deoxyribo-
nucleotide
After this addition, the enzyme moves
back by one nucleotide distance
It checks the new base for correctness of
base-pairing

43. If the base-pairing is correct, the enzyme
moves ahead
It adds another deoxyribonucleotide
The replication continues

44. If a wrong base is detected, the last
nucleotide is split off
The splitting is catalysed by 3’  5’ exo-
nuclease activity of the enzyme
The correct nucleotide is, then, added by
the polymerase activity
This ensures a high fidelity in replication

45. Template
strand
New
strand
Mismatched
base
3’ 5’
Exonuclease
Polymerase
 

46. The ribonucleotides of RNA primer are
removed and replaced with deoxyribo-
nucleotides by DNA polymerase I
DNA polymerase I possesses three
different catalytic activities:
5’3’ Exonuclease activity
Polymerase activity
3’ 5’ Exonuclease activity

47. 5’  3’ Exonuclease activity removes the
last ribonucleotide from 5’-end of primer
In its place, a deoxyribonucleotide is
added by the polymerase activity
3’  5’ Exonuclease activity checks
correctness of base-pairing and removes
wrong nucleotides

48. The DNA fragments are joined together by
DNA ligase
Template strand and the new strand are
wound around each other by DNA gyrase
DNA polymerase II is a DNA repair
enzyme

49. The overall process of replication is
similar to that in prokaryotes
Many enzyme and protein factors involved
in replication are identical
But there are some distinct differences
Eukaryotic replication

50. The differences have
arisen due to:
Bigger size of DNA
More intricate cellular architecture
More complex cell biology in eukaryotes

51. Eukaryotic DNA is much bigger than pro-
karyotic DNA
For example, human genome is 800 times
as big as the E. coli genome
If there is only one site of origin, repli-
cation of human DNA will take a long time
Replication bubbles

52. For speedy replication:
Eukaryotic DNA has several sites of
origin
The template DNA is nicked at a
number of places
DNA is unwound at several places
Replication begins simultaneously at
these sites

53. EMB-RCG
The unwound portions form a number
of replication bubbles which are
replicated simultaneously
Origin Origin
Replication
bubbles

54. Eukaryotes have more DNA polymerases
than prokaryotes
Some enzymes are unique to eukaryotes
Eukaryotic enzymes

55. The enzymes unique to
eukaryotes are:
• DNA polymerase a
• DNA polymerase b
• DNA polymerase g
• DNA polymerase d
• DNA polymerase e
• DNA topoisomerase II
• Telomerase

56. DNA polymerase a
DNA polymerase a synthesizes the
lagging strand, and possesses:
Primase activity
DNA polymerase activity
3’  5’ Exonuclease activity
in some species

57. DNA polymerase b is similar to DNA
polymerase II of prokaryotes
It is a DNA repair enzyme
DNA polymerase b

58. In eukaryotes, some DNA is present in
mitochondria also
DNA polymerase g is a mitochondrial
enzyme
It replicates mitochondrial DNA
DNA polymerase g

59. DNA polymerase d
DNA polymerase d synthesizes the leading
strand
It possesses:
DNA polymerase activity
3’  5’ Exonuclease activity

60. DNA polymerase e
DNA polymerase e is similar to DNA poly-
merase I of prokaryotes and possesses:
5’ 3’ Exonuclease activity
Polymerase activity
3’ 5’ Exonuclease activity

61. DNA topoisomerase II is similar to DNA
gyrase of prokaryotes
It winds the template strand and the new
strand around each other
DNA topoisomerase II

62. The ends of chromosomes are known as
telomeres
Telomerase is required for replication of
telomeres
Telomerase

63. Telomere is a repeating sequence of
bases (GGGTTA)
Telomere is present at the ends of
eukaryotic chromosomes
The human telomere can have a length of
100-15,000 base pairs
Telomere

64. DNA polymerase can add nucleotides
only to an oligonucleotide
When replication machinery reaches the
telomere, bases left in the template strand
are too few to form the RNA primer
The end replication problem in
eukaryotes

65. DNA polymerase
cannot join these
No place
for a primer
∕
∕

66. Therefore, the terminal part of lagging
strand cannot be replicated
Since the terminal part is not replicated,
the telomere becomes shorter
This happens at the time of every cell
division

67. Each time a cell divides, some telomere is
lost
When the length of telomere decreases
below a critical limit, DNA cannot be
replicated
The cell undergoes apoptosis

68. Telomere shortening can be prevented by
adding some nucleotides to the telomere
The nucleotides are added at the 3’-end of
the template strand
As a result, the 3’-end becomes long
enough for primer synthesis
The solution

69. Addition of nucleotides requires a special
polymerase and a template
Polymerase activity and the template are
present in a single conjugated protein
The conjugated protein is telomerase

70. Telomerase is also known as telomere
terminal transferase
It is made up of a protein and a RNA
The protein part possesses polymerase
activity
The RNA component acts as a template
Telomerase

71. A few bases of the RNA component
hybridise with complementary bases of
telomere
The remaining part of RNA acts as a
template
The polymerase activity adds some
nucleotides to the 3’-end

72. Telomerase adds complementary deoxyribo-
nucleotides to the 3’-end of the telomere
----TTA3’ End of telomere
RNA template AAUCCCAAU
----TTAGGGTTA
AAUCCCAAU
3’ End of telomere
RNA template
Telomerase

73. Telomerase dissociates and translocates
towards the new 3’-end
RNA component binds to the new 3’-end,
and the telomere is extended again
This cycle is repeated several times

74. −−−TTAGGGTTA
Telomerase binding
AAUCCCAAU
−−−TTAGGGTTAGGGTTA
AAUCCCAAU
−−−TTAGGGTTAGGGTTA
Telomere extension
3’ End of telomere
Telomerase translocation
AAUCCCAAU
−−−TTAGGGTTATelomere end
Telomere end
Telomere end

75. When the telomere becomes sufficiently
long, the telomerase leaves it
A primer is synthesized opposite the 3’-
end of telomere
Deoxyribonucleotides are added to the
primer in the usual way
The end of the telomere is replicated

76. During foetal life, telomerase is present in
all the cells
After birth, it is present only in germ cells
Telomerase activity is lost in somatic cells
during development

77. But telomerase activity returns in cancer
cells
Therefore, there is no shortening of
telomeres in cancer cells
Cancer cells can go on dividing for ever

78. Replication of DNA is vital for life; inhibition
of replication prevents cell division
Inhibitors of eukaryotic replication can be
used as anti-cancer drugs
They will check the multiplication of cancer
cells
Inhibitors of replication

79. Cisplatin produces intra-strand purine
cross-links and prevents replication
Mitomycin C produces intra-strand G−G
and inter-strand C−G cross-links, and
blocks replication
Daunorubicin intercalates in DNA and
prevents access to DNA polymerase

80. Daunorubicin
Daunorubicin
Daunorubicin intercalation
DNA

81. Inhibitors acting selectively in prokaryotes
can be used as antibiotics
Antibiotics exploit the differences in pro-
karyotic and eukaryotic machinery
If a compound inhibits a prokaryotic
enzyme but not its eukaryotic counterpart,
it can be used as an antibiotic

82. Floxacin family of drugs (e.g. norfloxacin,
ciprofloxacin etc) inhibits DNA gyrase
DNA gyrase is present in prokaryotes only
The corresponding human enzyme is
DNA topo-isomerase II

83. The floxacins inhibit DNA gyrase but not
DNA topo-isomerase II
Being selective inhibitors of prokaryotic
replication, floxacins are used as antibiotics
Some other inhibitors of DNA gyrase are
novobiocin and nalidixic acid

84. Baltimore Temin Dulbecco
Reverse transcription
They named it as RNA-dependent DNA
polymerase
Baltimore, Temin and Dulbecco dis-
covered an unusual DNA polymerase

85. RNA-dependent DNA polymerase is
present in some RNA viruses
This enzyme can synthesize DNA using
RNA as a template
When such a virus infects a cell, the viral
enzyme synthesizes a DNA strand using
its own RNA as a template

86. Using the new DNA strand as a template,
a second strand of DNA is synthesized
The two strands wind around each other
forming a double-stranded DNA
This double-stranded DNA is a copy of the
viral RNA genome

87. The DNA copy of viral genome is incor-
porated in the genome of the infected cell
Incorporation is catalysed by integrase, a
viral enzyme
Thus, viral genome becomes an integral
part of host cell genome

88. EMB-RCG

89. The infected cell begins to transcribe and
translate viral genes
Viral proteins are synthesized by the
infected cell
The proteins surround the RNA transcripts
of the viral genome
This results in the formation of new
viruses

90. RNA-dependent DNA polymerase catalyses
reverse of the normal transcription
Hence, it is also known as reverse trans-
criptase
Viruses possessing reverse transcriptase are
known as retroviruses

91. Human immunodeficiency virus (HIV) is
an example of a retrovirus
HIV infects helper T cells of human beings
and cripples the immune system
Reverse transcriptase is used as a tool in
recombinant DNA technology