Updated : Define Replication, its criteria, Requirements for replication, related terminology, Steps and importance of replication

1. Replication
Dr Ifat Ara Begum
Associate Professor
Department of Biochemistry
Dhaka Medical College Dhaka
24/11/2020

2. Structure of DNA
2

3. 3

4. 4
Chargaff's rule”
A = T & C = G

5. 5

6. What is Replication?
 Fundamental process by which cell copies its DNA to transfer the genetic
information to daughter cells
 DNA directed DNA synthesis where the base sequence of daughter DNA (newly
synthesized DNA) is identical to the base sequence of parent DNA (template).
 The major event in S-phase of cell cycle
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7. 7
Principle / Molecular mechanism of
Replication

8. 8
Replication is the basis
for the biological
inheritance
Precise and accurate DNA replication is
necessary to prevent genetic abnormalities
which often lead to cell death or disease
Cell must replicate its
DNA before division
Ultimately, exactly two identical
semi-conserved chromosomes are
produced

9. # DNA template
# Primer (free 3'-OH group):
Short RNA segment having 10 nucleotides or a preexisting cellular
DNA fragment
# Magnesium ion
# Activated deoxy nucleoside tri-
phosphate (dNTP):
dATP, dGTP, dCTP, dTTP
# DNA Polymerase
# DNAP associated proteins and
enzymes
Primase, Helicase, SSBP, DNA
Ligase, Topoisomerase (DNA gyrase)
Requirements for Replication

10. Enzyme & Protein Function
DNAP
(DNA directed DNAP)
# Catalyzes the formation of ester bonds between nucleotides
# Polymerization of dNTP to synthesize DNA with proof reading
Primase Synthesis of RNA Primer
Helicase Unwinding / melting of DS DNA in to two single strands
Single Strand Binding
Protein
(SSBP)
Prevention of premature reassociation / annealing of melted DNA
DNA ligase Seals the single strand nick & connects the okazaki fragments on lagging
strand
Topoisomerase / DNA
Gyrase
Relieves torsional strain resulting from helicase induced unwinding
(Helps to overcome topological crisis)

11. Criteria / Features of Replication
11
Semi conservative process: 50% of parental DNA is
conserved in each daughter DNA
Symmetric process: After melting of DS parental DNA, each of the two SS
parental DNA serves as template on which new complementary DNA is
synthesized
-Template is copied always from 3' to 5' direction & new strand is
synthesized from 5' to 3' direction
- Needs primer

12. 12
Bidirectional process: From a specific site (ORI),
replication simultaneously proceeds in both direction
- Non selective process: The whole genome is copied, not a selected segment
- Semi discontinuous process: Between two strands, one (leading / forward strand) replicates
continuously uninterrupted whereas the other one (lagging / retrograde strand) replicated
discontinuously with interruption
- Process of high fidelity : Because, DNAP has proof reading (3' to 5' exonuclease) property, it
can recognize & remove any mismatched nucleotide recruited during synthesis of daughter DNA
strand
- No need of post replicational modification

13. Semi discontinuous DNA
Replication

14. Energy of replication
 The nucleotides used for replication arrives as nucleoside
tri phosphate
i.e.
the bases are with their own energy source for bonding
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15. 15
Some related topics

16. Enzymes that catalyze the synthesis
of DNA molecules from nucleoside
triphosphates (deoxyribonucleotides),
the molecular precursors / building
blocks of DNA.
They usually work in pairs & read the
existing (template) DNA strand to create
2 new strands that match the existing
one (proof reading)
They are essential for DNA replication
They can add free nucleotides only to the 3'
end of the growing / newly forming DNA
strand.
So, they need a starter nucleotide to make a
bond.
(so, they need primer)
DNA Polymerase

19. TYPE FUNCTION
DNAP-α  Synthesis of primer
Initiation of DNA synthesis
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

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Origin of Replication
(ORI):
 It is the place in DNA double
helix which unwinds first to
initiate replication
 It is identified by consensus
sequence rich in AT bp
 Replication begins at multiple
ORI in eukaryotes and proceeds
bidirectionally

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22. 22
Replicon:
 A region of DNA
that replicates from a single ORI
Or
Replicated region of DNA
centering a definite ORI
 Each replicon has origin,
terminus and control elements of
replication

23. 23
Replication bubble :
 An unwound and open region of a
DNA helix where
DNA replication occurs.
[ Helicase unwinds only a small section
of the DNA at a time in a place called
the ORI ]
Replication Fork :
 The two sides of each bubble where it goes from zipped to
unzipped are called replication forks
 i.e. 2 R. forks per R. bubble
 Structurally, it consists of Helicase, SSBP, Primase, DNAP

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Topological Crisis:
 It is created when melting of DS
DNA applies turning force creating
positive super coiling / positive super
twisting forward to the unmelted
DNA double helix.
 Cessation of further DNA separation
occurs due to torsional strain.
 Topoisomerase (DNA gyrase)
relieves the torsional strain resulting
from helicase-induced unwinding

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Primer:
 A primer is a short nucleic acid
sequence that provides a starting
point for DNA synthesis.
 In living organisms, it may be a
short RNA segment having 10
nucleotides / the preexisting cellular
DNA fragment
 Before DNA replication can occur,
primer is synthesized by an enzyme
called primase, which is a type of
RNA polymerase
 The synthesis of a primer is necessary
because DNAP can attach new DNA
nucleotides only to an existing strand of
nucleotides. As such, primer serves to
prime and lay a foundation for DNA
synthesis.
i.e. Primer serves as a starter sequence for
DNAP

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 For leading strand: Only one RNA primer is required .
 For lagging strand: Its number depends on the number of Okazaki fragments
 The primers are removed before DNA replication is complete and the gaps in the
sequence are filled in with DNA by DNA polymerases

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 In the laboratory, scientists can design and synthesize DNA primers
with specific sequences that bind to sequences in a single-stranded DNA
molecule.
 These DNA primers are commonly used to perform the polymerase
chain reaction to copy pieces of DNA or for DNA sequencing.

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Leading Strand & Lagging
Strand:
 In replication, both the strands of DNA act as
template to synthesize their corresponding
complementary strands
 Unwinding / melting of parent DS DNA provides two
SS parent DNA to act as template

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Binding of RNA primer with
template follows the basic rule of
anti parallelism
i.e.
5' end and 3' end of template will
be just opposite to the 3' end and
5' end of primer respectively
Another basic rule to follow is :
To synthesize new DNA strand,
primer grows only in 5' to 3'
direction
reading the template in 3' to 5'
direction only

31.  As DNAP can add nucleotides only from 5' to 3' direction, synthesis of one strand is
continuous where replication fork moves in 3' to 5' direction.
This is leading strand
 Here, Primer binds at the proximal end of replication bubble and daughter DNA strand
grows distally and continuously

32.  As DNAP can add nucleotides only from 5' to 3' direction, synthesis of other strand is not
continuous where replication fork moves in 5' to 3' direction.
 The synthesis then proceeds in short segments (okazaki fragments) in the 5' to 3' direction
This is lagging strand
 Here, primase comes into action repeatedly to make primer that gets attached at the distal
end of replication bubble & then synthesizes complementary DNA fragments proximally

33. Features Leading strand Lagging strand
Replication fork
movement
From 3' to 5' direction From 5' to 3' direction
Binding of primer At proximal end of replication
bubble
At distal end of replication
bubble
Primer attachment Single Repeated
Daughter DNA
synthesis
Continuous & distally from ORI Discontinuous & proximally
towards ORI

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Okazaki Fragments:
 Pieces of replicated DNA produced on
the lagging strand during replication
 They are produced due to repeated
primer attachment with its template
strand
i.e.
Starting of synthesis of each okazaki
fragment needs a new RNA primer
 DNA ligase is required for joining
okazaki fragments at the end of
replication

35. Steps
of Replication

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

37. 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

38. 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
Joining of okazaki fragments DNA Ligase (requires
NAD)
DNA Ligase (requires
ATP)

39. Steps Prokaryotic Eukaryotic
Solving of topological
crisis
DNA topoisomerase DNA topoisomerase
Synthesis of telomeres Not required Telomerase

40. All these steps are described under three headings

41. Initiation:
 Identification of ORI
 Unwinding / melting of parent DS DNA to provide SS DNA
template (role of helicase)
 Formation of replication fork
 Synthesis of primer

42. Elongation:
 Attachment of primer with the template (SS parent DNA)
 Synthesis of new / daughter DNA complementary to template through
polymerization of dNTP by DNAP-δ & DNAP-ε.
 Solving of topological crisis by topoisomerase
 Excision of primer & its replacement by DNAP-β
 Sealing of nicks & joining of okazaki fragments by DNA ligase

43. Termination:
 Replication fork moves bidirectionally from the ORI until
adjacent replication fork fuse at opposite side when the
replication is completed

44. Others:
a. Synthesis of new histones
b. Reconstitution of chromatin structure with histones

45. Summary

46. Importance
of Replication

47.  It ensures the presence of complete complement of DNA to 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
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