DNA replication is the process by which DNA copies itself to produce identical daughter molecules. It occurs through a semi-conservative process in both prokaryotes and eukaryotes. In eukaryotes, replication is a complex multi-stage process involving initiation, formation of a pre-replication complex, conversion to an initiation complex, elongation via leading and lagging strand synthesis, and termination. Key proteins involved include ORC, Cdc6, Cdt1, MCM complexes, Cdc45, GINS, DNA polymerases, primase, ligase and topoisomerases. Replication occurs with high fidelity and is tightly regulated to prevent re-replication.

1. KrekarMardanMohammad

2. REPLICATION
It is a process in which the DNA copies itself to
produce identical daughter molecules of DNA.
It occurs only once in each cell.
It occurs very quick, accurate and at correct
time.
Replication of DNA occurs based on the
Chargaff’s Rule that is
 Cytosine – Guanine ( 3 H bonds)
 Adenine – Thymine (2 H bonds)

3. Modes of Replication
 Delbruck suggest that Watson-Crick model of DNA
could theoretically be replicated by three modes
 Conservative
 Semi-conservative
 dispersive

5. Simple Process
G1
•Replication initiated
S
•DNA synthesis occur
•Two daughter copies
are produced
G2
•Repair mechanisms
occur
Finally, one copy of the genomes is
segregated to each daughter cell at mitosis
or M phase.
These daughter copies each contain one
strand from the parental duplex DNA and
one nascent anti-parallel strand.
This process is conserved from
prokaryotes to eukaryotes and the
mechanism is called semi-conservative
mode of replication.

6. ComplexProcess
 DNA replication in eukaryotes divided into three stages
1. Initiation ( Formation of Pre – Replicative
Complex)
2. Initiation complex
3. Elongation (Replication fork and Polymerization)
4. Termination

7. Initiation of Replication
 It is the first step in eukaryotic replication in which most
of the proteins combines to form Pre – Replicative
complex (Pre-RC).
 Involved proteins
 Origin Recognition complex (ORC)
 Cell division cycle 6( Cdc 6)
 Chromatin licensing and DNA Replication factor 1( Cdt 1)
 Minichromosome Maintenance Protein Complex (Mcm 2-
7)

8. Steps in initiation
ORC binds in the
Ori-c Site of the
DNA
Recruits the Cdc 6
Cdc 6 Binds with
ORC in ATP
dependent manner
Cdc 6 recruits the
Cdt 1
Cdt 1 is required for
licensing the
chromatin for
Replication
Cdt 1 binds with C
terminus of Cdc 6
Finally binding all
three protein recruit
Mcm
Mcm finally binds
with Chromatin
These following
steps occur in G1
phase of cell cycle

10.  The activity of Cdt 1 during the cell cycle is regulated by a
protein called Geminin.
 It also inhibits Cdt 1 activity during the S phase in order to
prevent the re-replication of DNA, Ubiquitination and
proteolysis.

11. Functions of Mcm Complex
 Minichromosome Maintenance Complex has helicase
activity and inactivation of any of the six protein will
prevent the progress of formation of replication fork.
 It also has ATPase activity. A mutation at any one of the
Mcm protein complex will reduce conserved ATP
binding site.
 Mcm complex is a hexamer with Mcm 3, Mcm 7, Mcm
2, Mcm 6, Mcm 4, Mcm 5.

12. Initiation Complex
 It is the 2nd stage in DNA replication where the Pre –
Replicative complex is converted into Initiation complex.
 Involved proteins
 Cell Division Cycle 45 ( Cdc 45)
 GINS
 Cyclin Dependent Kinase ( CDK)
 Dbf 4 Dependent Kinase (DDK) – Combination of Cdc 7
and dbf 4

13. Steps in initiation complex
 Cdc 45 protein is a compound which is need for the
conversion of Pre – RC into initiation complex.
 Its binds with chromatin after the beginning of initiation
in late G1 phase by physically associated with Mcm 5.
 The binding of Cdc 45 is based on Clb - Cdc 28 as well as
the function of Cdc 6 and Mcm.
 GINS are essential for interaction of Mcm and Cdc 45 at
Ori-c site during initiation.

14.  GINS complex is composed of four small proteins namely
 Sld5 (Cdc105)
 Psf1 (Cdc101)
 Psf2 (Cdc102)
 Psf3 (Cdc103)
GINS represents 'go, ichi, ni, san' which means '5, 1, 2, 3' in
Japanese.

15.  At the onset of S phase, the pre-replicative complex must be
activated by two S phase-specific kinases in order to form an
initiation complex at an origin of replication.
 One kinase is the Cdc7-Dbf4 kinase called Dbf4-dependent
kinase (DDK) and the other is cyclin-dependent kinase (CDK).
 The CDK-dependent phosphorylation of Cdc6 has been
considered to be required for entry into the S phase.
 DDK targets the Mcm complex, and its phosphorylation leads
to the possible activation of Mcm helicase activity.

16. Elongation
 Once the initiation complex is formed and the cells pass
into the S phase, the complex then becomes a replisome
and elongation is initiated.
 Once the elongation is initiated, it form the replication
fork by unwinding the DNA strand.
 As the double helix of DNA separates from one side and
super coils are formed on the other side.
 The problem of super coils comes in the way of DNA
replication is solved by a group of enzymes called DNA
topoisomerase.

17. Replication Fork
 The replication fork is the junction the between the
newly separated template strands, known as the leading
and lagging strands, and the double stranded DNA.
 Elongation occur in 5’ to 3’ direction in both the leading
and lagging strand.

19. Leading Strand
 The leading strand is the template strand that is being
replicated in the same direction as the movement of the
replication fork.
 Nucleotides are added by the DNA Polymerase ε.
 DNA polymerase requires the RNA primer produced by
Primase.
 Elongation take place in 5’ to 3’ direction.
 Finally the primer are removed by RNAse H and the gap
is sealed by the DNA Ligase 1.

20. Lagging Strand
 DNA replication on lagging strand is discontinuous and
elongation opposite direction to replication fork.
 Nucleotide are added by the DNA Polymerase δ.
 Lagging strand used more RNA Primer for loading
nucleotide.
 DNA polymerase will synthesize short fragments of DNA
called Okazaki fragments which are added to the 3' end
of the primer. These fragments can be anywhere between
100-400 nucleotides long in eukaryotes.

21. Step 1 = Binding
Step 3 = Translocation
The binding-
polymerization-
translocation cycle can
occurs many times
This greatly lengthens
one of the strands
The complementary
strand is made by primase,
DNA polymerase and ligase
RNA primer
Step 2 = Polymerization

22. MERCi