V I G N E S H M
I M . S C . , H G M B
DNA Replication in Eukaryotes
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
Replication of DNA occurs based on the
Chargaff’s Rule that is
Cytosine – Guanine ( 3 H bonds)
Adenine – Thymine (2 H bonds)
Modes of Replication
Delbruck suggest that Watson-Crick model of DNA
could theoretically be replicated by three modes
• Replication initiated
• DNA synthesis
• Two daughter
copies are produced
• Repair mechanisms
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
This process is conserved from
prokaryotes to eukaryotes and the
mechanism is called semi-conservative
mode of replication.
DNA replication in eukaryotes divided into three
1. Initiation ( Formation of Pre – Replicative
2. Initiation complex
3. Elongation (Replication fork and
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).
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)
Steps in initiation
ORC binds in
the Ori-c Site of
Recruits the Cdc
Cdc 6 Binds with
ORC in ATP
Cdc 6 recruits
the Cdt 1
Cdt 1 is required
for licensing the
Cdt 1 binds with
C terminus of
all three protein
steps occur in G1
phase of cell
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.
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
It also has ATPase activity. A mutation at any one of
the Mcm protein complex will reduce conserved ATP
Mcm complex is a hexamer with Mcm 3, Mcm 7,
Mcm 2, Mcm 6, Mcm 4, Mcm 5.
It is the 2nd stage in DNA replication where the Pre –
Replicative complex is converted into Initiation
Cell Division Cycle 45 ( Cdc 45)
Cyclin Dependent Kinase ( CDK)
Dbf 4 Dependent Kinase (DDK) – Combination of Cdc 7 and
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.
GINS complex is composed of four small proteins
GINS represents 'go, ichi, ni, san' which means '5, 1, 2, 3' in
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
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.
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
The replication fork is the junction the between the
newly separated template strands, known as the
leading and lagging strands, and the double stranded
Elongation occur in 5’ to 3’ direction in both the
leading and lagging 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
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.
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
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
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
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.
Step 1 = Binding
Step 3 = Translocation
translocation cycle can
occurs many times
This greatly lengthens
one of the strands
strand is made by primase,
DNA polymerase and ligase
Step 2 = Polymerization