This document describes the process of DNA replication in eukaryotes. It occurs in S phase of the cell cycle and involves three main stages: initiation, formation of the initiation complex, and elongation. Initiation requires the assembly of pre-replication complexes containing ORC, Cdc6, Cdt1 and MCM proteins. In S phase, Cdc45 and GINS are recruited to form the initiation complex. Elongation proceeds bidirectionally from replication forks, with leading strand synthesis continuous and lagging strand discontinuous via Okazaki fragments. Replication terminates at telomeres.

1. V I G N E S H M
I M . S C . , H G M B
DNA Replication in Eukaryotes

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. Complex Process
 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. DNA Polymerase

22. Termination
 The termination of replication in eukaryotic cells occures by
telomere regions and telomerase. Telomeres extend the 3' end of
the parental chromosome beyond the 5' end of the daughter
strand.
 This single-stranded DNA structure can act as an origin of
replication that recruits telomerase. Telomerase is a specialized
DNA polymerase that consists of multiple protein subunits and
an RNA component.
 The RNA component of telomerase anneals to the single
stranded 3' end of the template DNA and contains 1.5 copies of
the telomeric sequence. Telomerase contains a protein subunit
that is a reverse transcriptase called telomerase reverse
transcriptase or TERT. TERT synthesizes DNA until the end of
the template telomerase RNA and then disengages.

24. 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