Replication of DNA

1. REPLICATION
Dr. Farhana Atia
Assistant Professor
Department of Biochemistry
Nilphamari Medical College, Nilphamari
Email: farhana.atia@gmail.com

2. CELL CYCLE
Cell cycle refers to the
events between two mitotic
divisions.
4 phases
 G1 phase [gap1]
 Protein & RNA content
increased
 Duration- 12 hrs
 Synthetic phase [S]
 Replication of DNA (nuclear
DNA is completely replicated

3.  Gap 2 [G 2]
 Cytoplasmic
enlargement, DNA
repair & formation of
histone
 D : 4-5 hrs
 Mitotic [M]
 Cell division takes
place
 D : 1 h
Total cycle : 20-22 hrs

4.  After cell division
daughter cell either
enter into G₀ phase
[undeviding/ dormant] or
reenter the cell cycle
when growth & repair is
necessary.
 In a normal cell
population most of the
cells are in G₀ phase.

5. DNA Organization
 A typical human contain 46 chromosomes, in
which total DNA is approximately 1 meter long.
 Eukaryotic DNA is associated with tightly bound
basic protein called histone
 DNA & histone form a beads on a string
structure. This structural unite is called
nucleosome. This are further arranged into
increasingly more complex structures that
organize & condense the long DNA molecules
into chromosome.

6.  The complex of DNA & protein [H & nonH]
found inside the nuclei is called chromatin.
HISTONES
 5 classes
 H1, H2A, H2B, H3, H4
 Rich in basic amino acid
 So positively charged in normal pH
 Form ionic bond with negative charged DNA
DNA Organization contd

7. NUCLEOSOMES
 2 molecules each of H2A, H2B, H3, H4 form
the structural core of individual nucleosome
beads [octamer].
 Around this core a segment of DNA double
helix is wrapped nearly twice [1 & 3/4th turn]
forming a negatively super twisted helix.
 Neighboring nucleosomes are joined by linker
DNA
DNA Organization contd

9. H1
 Quite distinct form of histone- larger, more
basic, most tissue specific & species specific
 Not formed in nucleosome core
 Bind to the linker DNA chain between the
nucleosome beads.
 Facilitate the packing of nucleosome compact
structure.
DNA Organization contd

10. Polynucleosome
 Nucleosome can be packed more tightly to
form polynucleosome.
 This structure assume coil shape, often
referred to as nucleofilament
 These are organized into loops & anchored
to scaffold protein leads to final chromosomal
structure.
DNA Organization contd

12. REPLICATION
 It is the process in which DNA copies itself
to produce identical daughter molecules of
DNA.
 Process of DNA directed DNA synthesis.
 Purpose
Transmission of genetic information from
parent cell to daughter cell.

13. Requirements
 Activated deoxy neucleotide tri-phosphate [d-NTP]
 d ATP, d GTP, d CTP, d TTP
 DNA template
 DNA strands that will dictate step by step
polymerization of Ntide according to its base
sequence & base pairing rule
 [DNA]n + dNTP  [DNA]n+1 + Ppi
 PPi  ATP
 Primer
 Pre-existing DNA/ RNA segment provides free
3’OH to which Ntides are added.

14. Requirements
 Enzymes
 DNA polymerase : Polymerization of
deoxynucleotide.
 Helicases : unwinding of DNA
 DNA primase : Initiate synthesis of RNA primer
 Topoisomerase : Prevent supercoiling
 Single stranded binding protein [SSB] : Prevent
premature anealing of ds DNA
 DNA ligase : Seal the ss nick [okazaki frgmnt]
btween the nascent chain & newly formed chain
on lagging strand.

15. Formation of super coils

16. DNA polymerase
A family of enzyme. Synthesizes a new strand of
DNA by extending the 3' end of an existing Ntide
chain, adding new NT matched to the template
strand one at a time via the creation of
phosphodiester bonds.
 α (I): Contain primase, initiate DNA synthesis in both
leading & lagging strand.
 β : DNA repair.
 γ : Mitochondrial DNA replication.
 δ (III) : Replication of leading strand + proof reading
 ε (II): Replication of lagging strand + proof reading

17. General Features
 Occurs in both eukaryotic
& prokaryotic cells
 Semi-conservative
process: ½ parental DNA +
½ new DNA [ entire parent
conservation not possible]
 Symmetric process : Both
strands act as template at
the same time.
 Uni/ bidirectional

18.  Needs primer
 Template always copied
from 3’5’ direction &
chain grows from 5’3’
direction
 No need of post
replication modification
 Proof reading by
polymerase is a
process of high fidelity.

19. RNA primer
 DNA polymerase can not initiate synthesis. They
require RNA primer
 It is a short [10 NT] double stranded region
consists of RNA base paired to the DNA template
with a free OH group on 3’ end of RNA strand
 Free OH group serve as 1st acceptor of a NT by
DNA polymerase.
 Only one RNA Primer-required for the leading
strand
 RNA Primers for the lagging strand depend on
the number of “OKAZAKI FRAGMENTS”

20. Steps of DNA replication in eukaryotes
1. Identification of origins of replication
2. Unwinding [denaturation] of ds DNA to
provide a ss DNA template
3. Formation of replication fork; synthesis of
RNA primer
4. Initiation of DNA synthesis & elongation
5. Formation of replication bubbles with
ligation of this newly synthesized DNA
segments

21. Steps
 Identification of origin of replication (ori)
by a particular dna-A protein
Origin of replication : DNA replication begins
at a single unique NT sequence. This site is
called ori.

22.  Unwinding [denaturation] of ds DNA to provide
a ss DNA template by helicase.
 Formation of replication fork- ds unwind &
separate to form V where active synthesis
occur. It consists-
1. DNA helicase
2. A primase- initiate synthesis of an RNA molecule
for priming DNA synthesis
3. DNA polymerase- initiates nascent, daughter
strand synthesis
4. SSBs- bind to ssDNA & prevent premature
Steps

23. Replication Fork

24.  Initiation of DNA synthesis & elongation
according to RNA primer by primase. RNA
primer is complementary & antiparallel to the
DNA template.
 In leading strand- continuous & towards
replication fork by DNA polymerase.
 In lagging strands- Discontinuous as ‘okazaki
fragments’ & away from replication fork.
 In both strands synthesis is 5’3’ direction
 Then RNA pieces are removed.
Steps

25.  Formation of replication bubbles with ligation of
this newly synthesized DNA segment by ligase.
Proof reading- by DNA polymerase.
 Reconstitution of chromatin structure by
organizing DNA & histone
 Parental histone octamers are conserved &
remain associated with only one of the parental
strand of DNA
 Synthesis of new histone occurs simultaneously
with DNA replication
 These are associated with only one of new
Steps

26. Replication Bubbles

27. DNA repair is a collection of processes by which a
cell identifies and corrects damage to the DNA
molecules that encode its genome.
 Despite of elaborate proof reading system
employed in DNA synthesis some mismatches can
occur like incorrect base pairing or insertion of
one-few extra Ntide.
 DNA is constantly being subjected to
environmental insult that causes the alteration or
removal of NT base.
 Cell possesses an inbuilt system to repair the
DNA REPAIR

28. Types of DNA Damage
 Single base alteration
Depurination
Deamination of CU/ Ahypoxanthine
Insertion of deletion of Ntide
 2 base alteration
UV light induced T-T dimer
 Double strand/chain break
Ionizing radiation
Radioactive disintegration of backbone element
Oxidative free radical formation
 Cross linkage
Between bases of same/ opposite strand

29. Major mechanism of DNA repair
Base excision repair (BER)
Ntide excision repair (NER)
Mismatch repair (MR)
Homologous recombination (HR)
Nonhomologous end-joining (NHEJ)