DNA
REPLICATION
DNA Replication:
DNA replication is the process by which a double-
stranded DNA molecule is copied to produce two identical dna
molecule. Replication is an essential process because, whenever
a cell divides, the two new daughter cells must contain the same
genetic information, or DNA, as the parent cell.
Biological significance
Extreme accuracy of DNA replication is necessary in order to preserve
the integrity of the genome in successive generations
What are the basic rules of replication?
Semi-conservative
Starts at the ‘origin’
Synthesis always in the 5-3’ direction
Can be uni or bidirectional
Semi-discontinuous
RNA primers required
DNA replication has 3 models
Semi-conservative
replication:
• One strand of duplex
passed on unchanged to
each of the daughter cells.
• This 'conserved' strand
acts as a template for the
synthesis of a new,
complementary strand by
the enzyme DNA
polymerase
How do we know that DNA replication is semiconservative?
Meselson-Stahl experiments
Starts at origin
• Initiator proteins identify specific base of DNA
called sites of origin
Prokaryotes
• Prokaryotes – single origin site E.g [Link] -
oriC
• Eukaryotes – multiple sites of origin
(replicator)
• E.g. yeast - ARS (autonomously replicating
sequences)
Eukaryotes
In what direction does DNA replication occur?
Synthesis is always in the 5’-3’ direction
What happens if a base mismatch
occurs?
Where does energy for addition
of nucleotide come from?
nucleotides are connected to ATP, CTP,
GTP, and TTP and when they are
connected with DNA polymerase, each
of those molecules drop off the part
consisting of the backbone and base
and the other two phosphate groups
just leave
Why does DNA replication only occur in the 5’ to 3’ direction?
DNA replication goes in the 5' to 3' direction because DNA polymerase acts on the 3'-OH of the existing strand for adding free nucleotides.
Uni or bidirectional
Replication forks move in one or opposite
directions
Semi-discontinuous replication
Anti parallel strands replicated
simultaneously
Because the leading strand is synthesized
continuously and the lagging strand is
synthesized discontinuously, the overall
process is described as semi-discontinuous.
Okazaki and colleagues obtained evidence
for discontinuous
Leading strand synthesis continuously in
5’– 3’
Lagging strand synthesis in
discontinuously in 5’-3’
Semi-discontinuous replication
New strand synthesis always in the 5’-3’ direction
Core proteins at the replication fork
Topoisomerases - Prevents torsion by DNA breaks
Helicases - separates 2 strands
Primase - RNA primer synthesis
Single strand - prevent reannealing
binding proteins of single strands
DNA polymerase - synthesis of new strand
Tethering protein - stabilises polymerase
DNA ligase - seals nick via phosphodiester linkage
What kind of enzyme synthesizes the new DNA strand?
1) RNA polymerase
2) DNA Polymerase
3) Primase
4) Helicase
5) Topoisomerase
The mechanism of DNA replication in prokaryotes
Initiation
During initiation, proteins bind to the origin
of replication while helicase unwinds the DNA helix
and two replication forks are formed at the origin
of replication.
Elongation
During elongation, a primer sequence is added with
complementary RNA nucleotides, which are then
replaced by DNA nucleotides.
Termination
Proteins release the replication complex
Initiation
Elongation
Termination of replication
The mechanism of DNA replication
[Link]
Initiation of Replication in eukaryotes
• 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)
Steps in initiation
ORC binds in the Finally binding all
Cdt 1 binds with C
Ori-c Site of the three protein recruit
terminus of Cdc 6
DNA Mcm
Cdt 1 is required for
licensing the Mcm finally binds
Recruits the Cdc 6
chromatin for with Chromatin
Replication
Cdc 6 Binds with These following steps
Cdc 6 recruits the
ORC in ATP occur in G1 phase of
Cdt 1
dependent manner cell cycle
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 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.
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
Elongation
• 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.
• Nucleotides are added by the DNA Polymerase ε.
• DNA polymerase requires the RNA primer produced by Primase.
• Finally the primer are removed by RNAse H and the gap is sealed
by the DNA Ligase 1.
Proteins of eukaryotes and prokaryotes
Termination
Eukaryotes lack termination sequences and proteins analogous to Ter sites and Tus protein in
prokaryotes
Eukaryotes initiate DNA replication at multiple points in the chromosome,
So replication forks meet and terminate at many points in the chromosome; these are not known
to be regulated in any particular manner.
Telomeric Replication
• Repair polymerases & ligase cannot fill gap
at end of chromosome after RNA primer is
removed
• If this gap is not filled, chromosomes would
become shorter each round of replication
Telomeres of linear chromosomes
Solutions
1. Eukaryotes have tandem repeated sequences at the ends of
their chromosomes
2. Telomerase (composed of protein and RNA primer
complementary to the telomere repeat) binds to the terminal
telomere repeat and catalyzes the addition of new repeats
Summary of replication
Eukaryotic chromosomes have multiple origins of replication
1. True
2. False
In what direction is the newly synthesized DNA produced?
1. 5'-3'
2. 3'-5'
3. In the direction of the major groove
4. Both 5'-3' and 3'-5' depending on which strand is being replicated
Nucleotides are always added to the growing DNA strand at the 3’ end, at which the DNA
has a free ______ on the 3’ carbon of its terminal deoxyribose.
1. Phosphate group
2. Hydroxyl group
3. Nitrogen base
4. Methyl group
What is the sequence (1 to 6) in which these proteins function during DNA replication?
____ RNA primase
____ DNA ligase
____ DNA polymerase
____ Topoisomerase
____ DNA helicase
____ tethering proteins
Why is an RNA primer necessary for DNA replication?
A. The RNA primer is necessary for the activity of DNA ligase.
B. The RNA primer creates the 5’ and 3’ ends of the strand.
C. DNA polymerase can only add nucleotides to RNA molecules.
D. DNA polymerase can only add nucleotides to an existing strand
