DNA replication is the process where DNA copies itself to produce identical daughter molecules. It occurs during the S phase of the cell cycle and involves unwinding of the DNA double helix, synthesis of new strands complementarity to each existing strand, and production of two identical copies of the original DNA molecule. DNA polymerase synthesizes new DNA strands in the 5' to 3' direction by adding nucleotides that are complementary to the template strand, requiring primers, nucleotides, and several enzymes. Errors can occur which are corrected by DNA repair mechanisms like base excision repair.

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)