Review on structure of DNA, detail on replication

1. REPLICATION
DR IFAT ARA BEGUM
Assistant Professor(Biochemistry)
Dhaka Medical College, Dhaka

2. REVIEW ON STRUCTURE OF
DNA
The DNA double helix comprises two
complementary polynucleotide strands
that run anti-parallel to each other.
i.e. one strand runs in the 5' → 3'
direction, while the other runs in an
anti-parallel direction of 3' → 5'.

3. CONTD

4. CONTD
The building blocks of a DNA are
molecules called nucleotides, that
consists of :
I. Deoxyribose sugar (a 5-carbon sugar)
II.Nitrogenous base attached to the sugar
III. Phosphate group.

5. CONTD
There are four types of nucleotide
molecules depending on the type of
nitrogenous base attached.
These four nucleotides (and their
respective nitrogenous bases) are:
I. Adenosine (Adenine)
II.Thymidine (Thymine)
III.Guanosine (Guanine)
IV.Cytidine (Cytosine)

6. CONTD

8. CONTD
Chargaff's rule”
A = T & C = G

9. INTRODUCTION TO
REPLICATION
Fundamental process by which cell
copies its DNA to transfer the
genetic information to daughter
cells
DNA directed DNA synthesis
Base sequence of daughter DNA
(newly synthesized DNA) is
identical to the base sequence of
parent (template) DNA

11. CONTD
The major event in S-phase of cell
cycle

12. CONTD
It is the basis for the biological
inheritance
Cell must replicate its DNA before
division
Ultimately, exactly two identical
semi-conserved chromosomes are
produced

13. CONTD
Precise and accurate DNA
replication is necessary to prevent
genetic abnormalities which often
lead to cell death or disease.

14. REQUIREMENTS
DNA template
Primer (free 3’-OH group):
short RNA segment having 10
nucleotides or a preexisting cellular
DNA fragment
Activated deoxy nucleoside tri-
phosphate (dNTP): dATP, dGTP, dCTP,
dTTP
Magnesium ion
Proteins & enzymes :
DNAP, SSBP, Primase, Helicase, Ligase,
DNA gyrase (topoisomerase)

15. FUNCTION OF
ENZYMES/PROTEIN
Unwinding/melting of
DS DNA in to single
strand

16. CONTD
Prevention of
premature
reassociation /
annealing of
melted DNA

17. CONTD
RNA primer synthesis

18. CONTD
Catalyzes joining of
nucleotides to 3’-
terminal of the growing
chain (deoxynucleotides
polymerization)

19. CONTD
Sealing the single
strand nick &
connecting okazaki
fragments on
lagging strand

20. CONTD
Relieves torsional
strain resulting from
helicase-induced unwinding

21. TOPOLOGICAL CRISIS
 It is created when melting of DS
DNA applies turning force creating
positive super coiling forward to
the unmelted DNA double helix.
 It tends to cause cessation of
further DNA separation due to
torsional strain.

23. CRITERIA/FEATURES
Semi conservative process: 50% of
parental DNA is conserved in each of
daughter DNA
Copying of template always occurs
from 3’ to 5’ direction & synthesis of
new strand from 5’ to 3’ direction
Needs primer

24. CONTD
Symmetric process: After unwinding
of DS DNA, each of the 2 single
stranded parental DNA serves as
template for synthesis of new
complementary daughter DNA
Bidirectional process: From a
specific ORI, replication proceeds in
both direction
Non selective process: The whole
genome is copied

25. CONTD
Semi discontinuous process: B/w 2
strands, one replicates continuously
without interruption whereas the
other one replicated discontinuously
with interruption.
Process of high fidelity : As DNAP
has proof reading property,
replication is a process of high
fidelity & there is no need of post
replicational modification

26. WHY CALLED SEMI
CONSERVATIVE?

27. WHY CALLED SEMI
DISCONTINUOUS?

28. DNA POLYMERASE
Enzymes that create DNA molecules
by assembling nucleotides, the
building block of DNA
These are essential to DNA
replication
They usually work in pairs & read
the existing (template) DNA strand to
create 2 new strands that match the
existing one (proof reading)

29. CONTD

30. CONTD
DNAP can only add nucleotides to 3’
end of a growing DNA strand.
So, they need a starter nucleotide to
make a bond. (so, they need primer)
Need to remember, the synthesis of
new strand occurs from 5’ to 3’
direction and copying of template
from 3’ to 5’ direction

32. CONTD
TYPE FUNCTION
DNAP-α •Initiation of DNA synthesis
•Synthesis of primer
DNAP-β •Excision of primer
•DNA repair
DNAP-γ Mitochondrial DNA replication with
proof reading
DNAP-δ Synthesis of lagging strand with
proof reading
DNAP-ε •Synthesis of leading strand with
proof reading
•DNA repair

33. ORI
Origin of replication
The place in DNA double helix
which unwinds first to initiate
replication
Identified by consensus sequence
rich in AT bp
Replication begins at multiple ORI
in eukaryotes and proceeds
bidirectionally.

37. REPLICON
Functional unit of replication
Replicated region of DNA centering
a definite ORI
Or
Space between 2 adjacent origins
Each replicon has origin, terminus
and control elements of replication

38. REPLICATION BUBBLE
AND REPLICATION
FORK
A replication bubble is an unwound
and open region of a DNA helix
where DNA replication occurs.
[Remember, helicase unwinds only a
small section of the DNA at a time in
a place called the origin of
replication]

39. CONTD
Like a zipper that has unzipped in the
middle, the separated DNA strands
form a little open pucker. This is the
replication bubble.

40. CONTD
The two sides of each bubble (where it
goes from zipped to unzipped) are
called replication forks.

41. SO, IN SHORT
At ORI, DNA strands separate
forming a replication bubble with
replication fork at each end.
And as from a definite ORI,
replication proceeds
bidirectionally, it forms Y-shaped
replication fork on either side of
replication bubble.

43. CONTD
Structurally, the replication fork
consists of:
 Helicase
 SSBP
 Primase
 DNAP

44. RNA PRIMER
Short RNA segment having 10
nucleotides / preexisting cellular DNA
fragment
Synthesized by primase
Serves as a starter sequence for DNAP
Only 1 RNA primer is required for
leading strand. For lagging strand, its
number depends on the number of
Okazaki fragments
RNA primer has a free 3’ OH group to
which the first nucleotide is bound

45. REPLICATION
The nucleotides used for replication
arrives as nucleoside tri phosphate
(i.e. the bases are with their own
energy source for bonding)

46. STRAND
In replication, both the strands of
DNA acts as template to synthesize
their corresponding complementary
strands
Unwinding of dsDNA provides 2
ssDNA to be used as template
There are 2 rules:
a)Rule of anti parallelism
b)Primer can grow only from 5’ to 3’
direction

47. CONTD
As DNAP can add nucleotides only
from 5’ to 3’ direction, synthesis in
one strand is continuous in the 5’ to
3’ direction towards the fork . This is
leading strand

48. CONTD
In the other strand, as the
replication fork opens, multiple sites
of initiation are exposed. The
synthesis then proceeds in short
segments (okazaki fragments) in the
5’-3’ direction. This is lagging strand

49. LEADING VS. LAGGING STRAND
Leading strand Lagging strand
Replicated strand of
DNA which grows
continuously without
any gap
Replicated strand of DNA
which is formed in short
segments (okazaki
fragments).
Its growth is continuous Its growth is
discontinuous
DNA ligase is not
required
DNA ligase is required
for joining okazaki
fragments

50. CONTD
Leading strand Lagging strand
Its template opens in 3’-
5’ direction
Its template opens in 5’-3’
direction
The direction of growth
of leading strand is 5’-3’
The direction of growth
of lagging strand is 3’-5’
but in okazaki fragment, it
is 5’-3’

51. CONTD
Leading strand Lagging strand
Only a single RNA
primer is required
Starting of each okazaki
fragment needs a new
RNA primer
Formation of leading
strand is quite rapid &
begins immediately at
the beginning of
replication
Formation of lagging
strand is slower & begins
a bit later than that of
leading strand

52. STEPS OF
REPLICATION
The process of DNA replication
comprises a set of carefully
orchestrated sequence of events to
duplicate the entire genetic content
of a cell.

53. Steps Prokaryotic Eukaryotic
Recognition of
ORI
dna-A protein unknown
Unwinding of
DNA double
helix
Helicase
(requires ATP)
Helicase
(requires ATP)
Stabilization of
unwound
template
strand
Single
stranded DNA
binding
protein (SSBP)
Single
stranded DNA
binding
protein (SSBP)
Synthesis of
RNA primer
Primase Primase

54. Steps Prokaryotic Eukaryotic
Synthesis of
DNA:
Leading strand
Lagging strand
(okazaki
fragments)
DNAP III
DNAP III
DNAP-ε
DNAP-δ
Removal of
RNA primers &
its replacement
with DNA
DNAP I DNAP-β

55. Steps Prokaryotic Eukaryotic
Joining of
okazaki
fragments
DNA Ligase
(requires
NAD)
DNA Ligase
(requires
ATP)
Solving of
topological
crisis
DNA
topoisomeras
e
DNA
topoisomeras
e
Synthesis of
telomeres
Not required Telomerase

56. ALL THESE STEPS ARE
DESCRIBED UNDER 3
HEADINGS:

57. A) INITIATION
 Identification of ORI
 Unwinding of a dsDNA to provide
a ssDNA template (role of helicase)
 Formation of replication fork
 Synthesis of primer

58. B) ELONGATION
 Attachment of primer with the
template (ssDNA)
 Synthesis of new / daughter DNA
complementary to template
through polymerization of dNTP
by DNAP-δ & DNAP-ε.

59. CONTD
 Solving of topological crisis by
topoisomerase
 Excision of primer & its
replacement by DNAP-β
 Sealing of nicks & joining of
okazaki fragments by DNA
ligase

60. C) TERMINATION
Replication fork moves
bidirectionally from the ORI until
adjacent replication fork fuse at
opposite side when the replication
is completed

62. D) OTHERS
a. Synthesis of new histones
b. Reconstitution of chromatin
structure with histones

63. SUMMARY

64. IMPORTANCE OF REPLICATION
It ensures the presence of
complete complement of DNA in
each daughter cell during cell
division, so that daughter cell
DNA becomes identical to that of
parent cell
It ensures duplication &
transmission of genetic
information from one generation
to next

65. DNA replication - 3D.mp4